4-substituted-1-(arylmethylidene)thiosemicarbazide, 4-substituted-1-(arylcarbonyl)thiosemicarbazide and analogs as activators of caspases and inducers of apoptosis and the use thereof

ABSTRACT

The present invention is directed to optionally substituted 4-substituted-1-(arylmethylidene)thiosemicarbazide, 4-substituted-1-(arylcarbonyl)thiosemicarbazide and analogs thereof, represented by the Formulae I and II:                    
     wherein A 1 , A 2 , Q and R 1 -R 3  are defined herein. The present invention also relates to the discovery that compounds having Formulae I and II are activators of caspases and inducers of apoptosis. Therefore, the activators of caspases and inducers of apoptosis of this invention may be used to induce cell death in a variety of clinical conditions in which uncontrolled growth and spread of abnormal cells occurs.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is in the field of medicinal chemistry. In particular,the invention relates to optionally substituted4-substituted-1-(arylmethylidene)-thiosemicarbazide,4-substituted-1-(arylcarbonyl)thiosemicarbazide and analogs, and thediscovery that these compounds are activators of caspases and inducersof apoptosis. The invention also relates to the use of these compoundsas therapeutically effective anti-cancer agents.

2. Related Art

Organisms eliminate unwanted cells by a process variously known asregulated cell death, programmed cell death, or apoptosis. Such celldeath occurs as a normal aspect of animal development, as well as intissue homeostasis and aging (Glucksmann, A., Biol. Rev. CambridgePhilos. Soc. 26:59-86 (1951); Glucksmann, A., Archives de Biologie76:419-437 (1965); Ellis, et al., Dev. 112:591-603 (1991); Vaux, et al.,Cell 76:777-779 (1994)). Apoptosis regulates cell number, facilitatesmorphogenesis, removes harmful or otherwise abnormal cells andeliminates cells that have already performed their function.Additionally, apoptosis occurs in response to various physiologicalstresses, such as hypoxia or ischemia (PCT published applicationWO96/20721).

There are a number of morphological changes shared by cells experiencingregulated cell death, including plasma and nuclear membrane blebbing,cell shrinkage (condensation of nucleoplasm and cytoplasm), organellerelocalization and compaction, chromatin condensation and production ofapoptotic bodies (membrane enclosed particles containing intracellularmaterial) (Orrenius, S., J. Internal Medicine 237:529-536(1995)).

Apoptosis is achieved through an endogenous mechanism of cellularsuicide (Wyllie, A. H., in Cell Death in Biology and Pathology, Bowenand Lockshin, eds., Chapman and Hall (1981), pp. 9-34). A cell activatesits internally encoded suicide program as a result of either internal orexternal signals. The suicide program is executed through the activationof a carefully regulated genetic program (Wyllie, et al., Int. Rev. Cyt.68:251 (1980); Ellis, et al., Ann. Rev. Cell Bio. 7:663 (1991)).Apoptotic cells and bodies are usually recognized and cleared byneighboring cells or macrophages before lysis. Because of this clearancemechanism, inflammation is not induced despite the clearance of greatnumbers of cells (Orrenius, S., J. Internal Medicine 237:529-536(1995)).

It has been found that a group of proteases are a key element inapoptosis (see, e.g., Thornberry, Chemistry and Biology 5:R97-R103(1998); Thornberry, British Med. Bull. 53:478-490 (1996)). Geneticstudies in the nematode Caenorhabditis elegans revealed that apoptoticcell death involves at least 14 genes, 2 of which are the pro-apoptotic(death-promoting) ced (for cell death abnormal) genes, ced-3 and ced-4.CED-3 is homologous to interleukin 1 beta-converting enzyme, a cysteineprotease, which is now called caspase-1. When these data were ultimatelyapplied to mammals, and upon further extensive investigation, it wasfound that the mammalian apoptosis system appears to involve a cascadeof caspases, or a system that behaves like a cascade of caspases. Atpresent, the caspase family of cysteine proteases comprises 14 differentmembers, and more may be discovered in the future. All known caspasesare synthesized as zymogens that require cleavage at an aspartyl residueprior to forming the active enzyme. Thus, caspases are capable ofactivating other caspases, in the manner of an amplifying cascade.

Apoptosis and caspases are thought to be crucial in the development ofcancer (Apoptosis and Cancer Chemotherapy, Hickman and Dive, eds.,Humana Press (1999)). There is mounting evidence that cancer cells,while containing caspases, lack parts of the molecular machinery thatactivates the caspase cascade. This makes the cancer cells lose theircapacity to undergo cellular suicide and the cells become cancerous. Inthe case of the apoptosis process, control points are known to existthat represent points for intervention leading to activation. Thesecontrol points include the CED-9-BCL-like and CED-3-ICE-like gene familyproducts, which are intrinsic proteins regulating the decision of a cellto survive or die and executing part of the cell death process itself,respectively (see, Schmitt, et al., Biochem. Cell. Biol. 75:301-314(1997)). BCL-like proteins include BCL-xL and BAX-alpha, which appear tofunction upstream of caspase activation. BCL-xL appears to preventactivation of the apoptotic protease cascade, whereas BAX-alphaaccelerates activation of the apoptotic protease cascade.

It has been shown that chemotherapeutic (anti-cancer) drugs can triggercancer cells to undergo suicide by activating the dormant caspasecascade. This may be a crucial aspect of the mode of action of most, ifnot all, known anticancer drugs (Los, et al., Blood 90:3118-3129 (1997);Friesen, et al., Nat. Med. 2:574 (1996)). The mechanism of action ofcurrent antineoplastic drugs frequently involves an attack at specificphases of the cell cycle. In brief, the cell cycle refers to the stagesthrough which cells normally progress during their lifetime. Normally,cells exist in a resting phase termed G₀. During multiplication, cellsprogress to a stage in which DNA synthesis occurs, termed S. Later, celldivision, or mitosis occurs, in a phase called M. Antineoplastic drugs,such as cytosine arabinoside, hydroxyurea, 6-mercaptopurine, andmethotrexate are S phase specific, whereas antineoplastic drugs, such asvincristine, vinblastine, and paclitaxel are M phase specific. Many slowgrowing tumors, e.g., colon cancers, exist primarily in the G₀ phase,whereas rapidly proliferating normal tissues, e.g., bone marrow, existprimarily in the S or M phase. Thus, a drug like 6-mercaptopurine cancause bone marrow toxicity while remaining ineffective for a slowgrowing tumor. Further aspects of the chemotherapy of neoplasticdiseases are known to those skilled in the art (See, e.g., Hardman, etal., eds., Goodman and Gilman's The Pharmacological Basis ofTherapeutics, Ninth Edition, McGraw-Hill, New York (1996), pp.1225-1287). Thus, it is clear that the possibility exists for theactivation of the caspase cascade, although the exact mechanisms fordoing so are not clear at this point. It is equally clear thatinsufficient activity of the caspase cascade and consequent apoptoticevents are implicated in various types of cancer. The development ofcaspase cascade activators and inducers of apoptosis is a highlydesirable goal in the development of therapeutically effectiveantineoplastic agents. Caspase cascade activators and inducers ofapoptosis may also be a desirable therapy in the elimination ofpathogens, such as HIV, Hepatitis C and other viral pathogens. The longlasting quiescence followed by a disease progression may be explained byanti-apoptotic mechanism of these pathognes leading to persistentcellular reservoirs of the virions. It has been reported that HIV-1infected T leukemia cells or peripheral blood mononuclear cells (PBMCs)underwent enhanced viral replication in the presence of caspaseinhibitor Z-VAD-fmk. Furthermore, Z-VAD-fmk also stimulated endogenousvirus production in activated PBMCs derived from HIV-1-infectedasymptomatic individuals (Chinnaiyan, A. et. al. Nature Medicine. 3:333.1997). Therefore, apoptosis may serve as a beneficial host mechanism tolimit HIV spread and new therapeutics using caspase/apoptosis activatorsmay be useful to clear viral reservoirs from the infected individuals.Similarly, HCV infection also triggers anti-apoptotic mechanisms toevade host's immune surveillance leading to viral persistence andhepatocarcinogenesis (Tai, D. I., et. al., Hepatology 3:656-64 (2000)).Therefore, apoptosis inducers may be useful as therapeutics for HCV andother infectious disease. Moreover, since autoimmune disease and certaindegenerative diseases also involve the proliferation of abnormal cells,therapeutic treatment for these diseases could also involve theenhancement of the apoptotic process through the administration ofappropriate caspase cascade activators and inducers of apoptosis.

SUMMARY OF THE INVENTION

The present invention is related to the discovery that optionallysubstituted 4-substituted-1-(arylmethylidene)thiosemicarbazide,4-substituted-1-(arylcarbonyl)thiosemicarbazide and analogs, asrepresented in Formulae I and II, are activators of the caspase cascadeand inducers of apoptosis. Thus, an aspect of the present invention isdirected to the use of compounds of Formulae I and II as inducers ofapoptosis.

The compounds of the present invention are represented by Formulae I andII:

or pharmaceutically acceptable salts or prodrugs thereof, wherein:

A₁ and A₂ each independently is optionally substituted and is aryl,heteroaryl, saturated carbocyclic, partially saturated carbocylic,saturated heterocyclic, partially saturated heterocyclic, arylalkyl, orheteroarylalkyl;

Q is S or O; and

R₁, R₂ and R₃ are independently hydrogen, optionally substituted alkylor optionally substituted cycloalkyl.

A second aspect of the present invention is to provide a method fortreating, preventing or ameliorating neoplasia and cancer byadministering a compound of one of the Formulae I and II to a mammal inneed of such treatment.

Many of the compounds within the scope of the present invention arenovel compounds. Therefore, a third aspect of the present invention isto provide novel compounds of Formulae I and II, and to also provide forthe use of these novel compounds for treating, preventing orameliorating neoplasia and cancer.

A fourth aspect of the present invention is to provide a pharmaceuticalcomposition useful for treating disorders responsive to the induction ofapoptosis, containing an effective amount of a compound of one of theFormulae I and II in admixture with one or more pharmaceuticallyacceptable carriers or diluents.

A fifth aspect of the present invention is directed to methods for thepreparation of novel compounds of Formulae I and II.

DETAILED DESCRIPTION OF THE INVENTION

The present invention arises out of the discovery that optionallysubstituted 4-substituted-1-(arylmethylidene)thiosemicarbazide,4-substituted-1-(arylcarbonyl)thiosemicarbazide and analogs, asrepresented in Formulae I and II, are potent and highly efficaciousactivators of the caspase cascade and inducers of apoptosis. Therefore,compounds of Formulae I and II arc useful for treating disordersresponsive to induction of apoptosis.

Specifically, compounds useful in this aspect of the present inventionare represented by Formulae I and II:

or pharmaceutically acceptable salts or prodrugs thereof, wherein:

A₁ and A₂ each independently is optionally substituted and is aryl,heteroaryl, saturated carbocyclic, partially saturated carbocylic,saturated heterocyclic, partially saturated heterocyclic, arylalkyl, orheteroarylalkyl;

Q is S or O; and

R₁, R₂ and R₃ are independently hydrogen, optionally substituted alkylor optionally substituted cycloalkyl.

Preferred compounds of Formulae I and II include compounds wherein A₁ isphenyl, naphthyl, pyridyl, quinolyl, isoquinolyl, thienyl, furyl,pyrrolyl or bicyclo[2.2.1]hept-5-en-2-yl; and wherein A₂ is phenyl,naphthyl, pyridyl, quinolyl, isoquinolyl, thienyl, furyl or pyrrolyl,each of which is optionally substituted. Preferably A₁ is phenyl,pyridyl or bicyclo[2.2.1]hept-5-en-2-yl; and A₂ is optionallysubstituted pyridyl or optionally substituted phenyl. Preferredcompounds of Formulae I and II also include compounds wherein R₁, R₂ andR₃ are hydrogen.

Preferably, the compounds useful in this aspect of the present inventionare represented by Formulae III to VI:

or a pharmaceutically acceptable salt or prodrug thereof, wherein:

A₂ is optionally substituted and is aryl, heteroaryl, saturatedcarbocyclic, partially saturated carbocylic, saturated heterocyclic,partially saturated heterocyclic, arylalkyl, or heteroarylalkyl;

Q is S or O; and

R₁, R₂ and R₃ are independently hydrogen, optionally substituted alkylor optionally substituted cycloalkyl.

R₄-R₈ are independently hydrogen, halo, haloalkyl, aryl, fused aryl,carbocyclic, a heterocyclic group, a heteroaryl group, alkyl, alkenyl,alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl,heteroarylalkenyl, heteroarylalkynyl, carbocycloalkyl, heterocycloalkyl,hydroxyalkyl, nitro, amino, cyano, acylamino, hydroxy, thiol, acyloxy,azido, alkoxy, aryloxy, heteroaryloxy, arylalkoxy, heteroarylalkoxy,haloalkoxy, carboxy, carbonylamido or alkylthiol, each of which isoptionally substituted.

Preferably R₁, R₂ and R₃ are hydrogen.

Preferably A₂ is optionally substituted and selected from the groupconsisting of cyclopentyl, phenyl, pyridinyl, furanyl, thiophenyl,pyrrolyl, pyrazolyl, imidazolyl, thiazolyl and 1,2,3-thiadiazolyl. Morepreferably A₂ is optionally substituted phenyl or optionally substitutedpyridinyl; most preferably, optionally substituted pyridinyl. Optionalsubstituents on A₂ include, without limitation, methyl, trifluoromethyl,methoxy, chloro, dimethylamino, nitro, hydroxy, fluoro, oxo, bromo,morpholino and pyrrolidino.

Preferably Q is S.

Exemplary preferred compounds that may be employed in the method of theinvention include, without limitation:

4-Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridin-2-ylmethylidene)thiomsemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(2-oxo-5-fluoroindol-3-ylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridine-4-carbonyl)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(cyclopentylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(1-methyl-2-(methoxycarbonyl)-pyrrol-4-ylmethylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-benzylidene-thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridin-4-ylmethylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyrrol-2-ylmethylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-hydroxybenzylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridin-3-ylmethylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(6-methyl-pyridin-2-ylmethylidene)thiosemicarbazide;

4-(3,5-Ditrifluoromethylphenyl)-1-(pyridin-2-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(pyridin-2-ylmethylidene)thiosemicarbazide;

4-(3-Trifluoromethylphenyl)-1-(pyridin-2-ylmethylidene)thiosemicarbazide;

4-(2,5-Dichlorophenyl)-1-(pyridin-2-ylmethylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridine-2-carbonyl)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-benzylidene-thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(6-methyl-pyridin-2-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-nitrobenzylidene)thiosemicarbazide;

4-(3-Methoxyphenyl)-1-(4-dimethylaminobenzoyl)thiosemicarbazide;

4-(2,5-Dichlorophenyl)-1-(2-chlorobenzoyl)thiosemicarbazide;

4-(2,5-Dichlorophenyl)-1-(4-dimethylaminobenzoyl)thiosemicarbazide;

4-(2,5-Dichlorophenyl)-1-(4-methoxybenzoyl)thiosemicarbazide;

4-(2,5-Dichlorophenyl)-1-(2-nitrobenzoyl)thiosemicarbazide;

4-(3-Chloro-4-fluorophenyl)-1-(2-methyl-6-trifluoromethyl-pyridine-3-carbonyl)thiosemicarbazide;

4-(2,6-Dichlorophenyl)-1-(3,5-dimethoxybenzoyl)thiosemicarbazide;

4-(2,5-Dichlorophenyl)-1-(3-chloro-benzo[b]thiophene-2-carbonyl)thiosemicarbazide;

4-(2,4,6-Trichlorophenyl)-1-(pyridine-3-carbonyl)thiosemicarbazide;

4-(3-Methylphenyl)-1-(3-methylbenzoyl)thiosemicarbazide;

4-(4-Chloro-2-methylphenyl)-1-(4-methoxybenzoyl)thiosemicarbazide;

4-(3-Methoxyphenyl)-1-(2-chlorobenzoyl)thiosemicarbazide;

4-(3-Thiomethoxyphenyl)-1-(2-nitrobenzoyl)thiosemicarbazide;

4-(3-Trifluoromethylphenyl)-1-(3,4,5-trimethoxybenwyl)thiosemicarbazide;

4-(4-Methoxyphenyl)-1-(4-(2,5-dimethylpyrrol-1-yl)benzoyl)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(cyclohexyl-methylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(3-carboxybenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-carboxybenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(4-carboxybenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(3-nitrobenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(3-chlorobenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-chlorobenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(3-hydroxybenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-hydroxybenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(4-dimethylaminobenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(furan-2-ylmethylidine)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(thiophen-2-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(5-nitrofuran-2-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(4,5-dimethylfuran-2-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(N-methylpyrrol-2-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(5-nitrothiophen-2-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(imidazol-2-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-methoxy-4-dimethylaminobenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(6-bromo-pyridin-2-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(imidazol-4-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(4-morpholino-3-nitrobenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(4-bromo-1H-pyrazol-5-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(4-chloro-1-methylpyrazol-3-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(1,2,3-thiazol-4-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(thiazol-2-ylethylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(thiazol-2-ylethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-furan-2-ylmethylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(3-chlorobenzylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(benzylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(4-pyrrolidinobenzylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(pyridin-2-ylmethylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-methoxy-4-dimethylaminobenzylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(4-chloro-1-methylpyrazol-3-ylmethylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(3-hydroxybenzylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-hydroxy-4-methoxybenzylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(1-methylpyrrol-2-ylmethylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(4,5-dimethylfuran-2-ylmethylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(thiophene-2-ylmethylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(1-methylimidazole-2-ylmethylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(6-methylpyridin-2-ylmethylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-hydroxy-4-methylbenzylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-hydroxy-5-chlorobeinzylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(2,4,5-trimethoxybenzylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(4-bromo-1H-pyrazol-5-ylmethylidne)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-methylthiazole-5carbonyl)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(4-chlorobenzoyl)semicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(2-methylthiazole-5-carbonyl)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-chlorobenzoyl)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(3-hydroxybenzoyl)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4,5-dimethylfuran-2-ylmethylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(2-methoxy-4-dimethylaminobenzylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(1-methylpyrrol-2-ylmethylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-pyrrolidinobenzylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(6-bromopyridin-2-ylmethylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(1,2,3-thiadiazol-4-ylmethylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-morpholino-3-nitrobenzylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-bromo-1H-pyrazol-5-ylmethylidne)thiosemicarbazide;and

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-chloro-1-methylpyrazol-3-ylmethylidene)thiosemicarbazide.

The present invention is also directed to novel compounds within thescope of Formulae I-VI. Exemplary novel compounds of this inventioninclude, without limitation:

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-benzylidene-thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridin-4-ylmethylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyrrol-2-ylmethylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-hydroxybenzylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridin-3-ylmethylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(6-methyl-pyridin-2-ylmethylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridine-2-carbonyl)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-benzylidene-thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(6-methyl-pyridin-2-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-nitrobenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(cyclohexyl-methylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(3-carboxybenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-carboxybenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(4-carboxybenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(3-nitrobenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(3-chlorobenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-chlorobenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(3-hydroxybenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-hydroxybenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(4-dimethylaminobenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(furan-2-ylmethylidine)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(thiophen-2-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(5-nitrofuran-2-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(4,5-dimethylfuran-2-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(N-methylpyrrol-2-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(5-nitrothiophen-2-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(imidazol-2-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-methoxy-4-dimethylaminobenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(6-bromo-pyridin-2-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(imidazol-4-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(4-morpholino-3-nitrobenzylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(4-bromo-1H-pyrazol-5-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(4-chloro-1-methylpyrazol-3-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(1,2,3-thiazol-4-ylmethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(thiazol-2-ylethylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(thiazol-2-ylethylidene)thiosemicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-furan-2-ylmethylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(3-chlorobenzylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(benzylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(4-pyrrolidinobenzylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(pyridin-2-ylmethylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-methoxy-4-dimethylaminobenzylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(4-chloro-1-methylpyrazol-3-ylmethylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(3-hydroxybenzylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-hydroxy-4-methoxybenzylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(1-methylpyrrol-2-ylmethylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(4,5-dimethylfuran-2-ylmethylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(thiophene-2-ylmethylidene)scmicarbazide;

4-(2,4-Dimethylphenyl)-1-(1-methylimidazole-2-ylmethylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(6-methylpyridin-2-ylmethylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-hydroxy-4-methylbenzylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-hydroxy-5-chlorobenzylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(2,4,5-trimethoxybenzylidene)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(4-bromo-1H-pyrazol-5-ylmethylidne)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(2-methylthiazole-5-carbonyl)semicarbazide;

4-(2,4-Dimethylphenyl)-1-(4-chlorobenzoyl)semicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(2-methylthiazole-5-carbonyl)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-chlorobenzoyl)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(3-hydroxybenzoyl)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4,5-dimethylfuran-2-ylmethylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(2-methoxy-4-dimethylaminobenzylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(1-methylpyrrol-2-ylmethylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-pyrrolidinobenzylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(6-bromopyridin-2-ylmethylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(1,2,3-thiadiazol-4-ylmethylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-morpholino-3-nitrobenzylidene)thiosemicarbazide;

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-bromo-1H-pyrazol-5-ylmethylidne)thiosemicarbazide;and

4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-chloro-1-methylpyrazol-3-ylmethylidene)thiosemicarbazide.

The term “alkyl” as employed herein by itself or as part of anothergroup refers to both straight and branched chain radicals of up to tencarbons. Useful alkyl groups include straight-chained and branched C₁₋₁₀alkyl groups, more preferably C₁₋₆ alkyl groups. Typical C₁₋₁₀ alkylgroups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl,tert-butyl, 3-pentyl, hexyl and octyl groups, which may be optionallysubstituted.

The term “alkenyl” as employed herein by itself or as part of anothergroup means a straight or branched chain radical of 2-10 carbon atoms,unless the chain length is limited thereto, including at least onedouble bond between two of the carbon atoms in the chain. Typicalalkenyl groups include ethenyl, 1-propenyl, 2-propenyl,2-methyl-1-propenyl, 1-butenyl and 2-butenyl.

The term “alkynyl” is used herein to mean a straight or branched chainradical of 2-10 carbon atoms, unless the chain length is limitedthereto, wherein there is at least one triple bond between two of thecarbon atoms in the chain. Typical alkynyl groups include ethynyl,1-propynyl, 1-methyl-2-propynyl, 2-propynyl, 1-butynyl and 2-butynyl.

Useful alkoxy groups include oxygen substituted by one of the C₁₋₁₀alkyl groups mentioned above, which may be optionally substituted.

Useful alkylthio groups include sulfur substituted by one of the C₁₋₁₀alkyl groups mentioned above, which may be optionally substituted. Alsoincluded are the sulfoxides and sulfones of such alkylthio groups.

Useful amino groups include —NH₂, —NHR₁₅ and —NR₁₅R₁₆, wherein is R₁₅and R₁₆ are C₁₋₁₀ alkyl or cycloalkyl groups, or R₁₅ and R₁₆ arecombined with the N to form a ring structure, such as a piperidine, orR₁₅ and R₁₆ are combined with the N and other group to form a ring, suchas a piperazine. The alkyl group may be optionally substituted.

Optional substituents on the alkyl, alkenyl, alkynyl, carbocyclic andheterocyclic groups include one or more halo, hydroxy, carboxyl, amino,nitro, cyano, C₁-C₆ acylamino, C₁-C₆ acyloxy, C₁-C₆ alkoxy, aryloxy,alkylthio, C₁-C₁₀ aryl, C₄-C₇ cycloalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₁-C₁₀ aryl(C₂-C₆)alkenyl, C₆-C₁₀ aryl(C₂-C₆)alkynyl, saturated andsaturated heterocyclic or heteroaryl.

Optional substituents on the aryl, aralkyl, aralkenyl, aralkynyl,heteroaryl and heteroarylalkyl groups include one or more halo, C₁-C₆haloalkyl, C₆-C₁₀ aryl, C₄-C₇ cycloalkyl, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₆-C₁₀ aryl(C₁-C₆)alkyl, C₆-C₁₀ aryl(C₂-C₆)alkenyl,C₆-C₁₀ aryl(C₂-C₆)alkynyl, C₁-C₆ hydroxyalkyl, nitro, amino, ureido,cyano, C₁-C₆ acylamino, hydroxy, thiol, C₁-C₆ acyloxy, azido, C₁-C₆alkoxy or carboxy.

The term “aryl” as employed herein by itself or as part of another grouprefers to monocyclic, bicyclic or tricyclic aromatic groups containingfrom 6 to 14 carbons in the ring portion.

Useful aryl groups include C₆₋₁₄ aryl, preferably C₆₋₁₀ aryl. TypicalC₆₋₁₄ aryl groups include phenyl, naphthyl, phenanthrenyl, anthracenyl,indenyl, azulenyl, biphenyl, biphenylenyl and fluorenyl groups.

Useful cycloalkyl groups are C₃₋₈ cycloalkyl. Typical cycloalkyl groupsinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl andcycloheptyl.

Useful saturated or partially saturated carbocyclic groups arecycloalkyl groups as described above, as well as cycloalkenyl groups,such as cyclopentenyl, cycloheptenyl and cyclooctenyl.

Useful halo or halogen groups include fluorine, chlorine, bromine andiodine.

The term “arylalkyl” is used herein to mean any of the above-mentionedC₁₋₁₀ alkyl groups substituted by any of the above-mentioned C₆₋₁₄ arylgroups. Preferably the arylakyl group is benzyl, phenethyl ornaphthylmethyl.

The term “arylalkenyl” is used herein to mean any of the above-mentionedC₂₋₁₀ alkenyl groups substituted by any of the above-mentioned C₁₋₁₄aryl groups.

The term “arylalkynyl” is used herein to mean any of the above-mentionedC₂₋₁₀ alkynyl groups substituted by any of the above-mentioned C₆₋₁₄aryl groups.

The term “aryloxy” is used herein to mean oxygen substituted by one ofthe above-mentioned C₆₋₁₄ aryl groups, which may be optionallysubstituted. Useful aryloxy groups include phenoxy and 4-methylphenoxy.

The term “arylalkoxy” is used herein to mean any of the above mentionedC₁₋₁₀ alkoxy groups substituted by any of the above-mentioned arylgroups, which may be optionally substituted. Useful arylalkoxy groupsinclude benzyloxy and phenethyloxy.

Useful haloalkyl groups include C₁₋₁₀ alkyl groups substituted by one ormore fluorine, chlorine, bromine or iodine atoms, e.g., fluoromethyl,difluoromethyl, trifluoromethyl, pentafluoroethyl, 1,1-difluoroethyl,chloromethyl, chlorofluoromethyl and trichloromethyl groups.

Useful acylamino (acylamido) groups are any C₁₋₆ acyl (alkanoyl)attached to an amino nitrogen, e.g., acetamido, chloroacetamido,propionamido, butanoylamido, pentanoylamido and hexanoylamido, as wellas aryl-substituted C₁₋₆ acylamino groups, e.g., benzoylamido, andpentafluorobenzoylamido.

Useful acyloxy groups are any C₁₋₆ acyl (alkanoyl) attached to an oxy(—O—) group, e.g., formyloxy, acetoxy, propionoyloxy, butanoyloxy,pentanoyloxy and hexanoyloxy.

The term heterocycle is used herein to mean a saturated or partiallysaturated 3-7 membered monocyclic, or 7-10 membered bicyclic ringsystem, which consists of carbon atoms and from one to four heteroatomsindependently selected from the group consisting of O, N, and S, whereinthe nitrogen and sulfur heteroatoms can be optionally oxidized, thenitrogen can be optionally quaternized, and including any bicyclic groupin which any of the above-defined heterocyclic rings is fused to abenzene ring, and wherein the heterocyclic ring can be substituted oncarbon or on a nitrogen atom if the resultant compound is stable.

Useful saturated or partially saturated heterocyclic groups includetetrahydrofuranyl, pyranyl, piperidinyl, piperazinyl, pyrrolidinyl,imidazolidinyl, imidazolinyl, indolinyl, isoindolinyl, quinuclidinyl,morpholinyl, isochromanyl, chromanyl, pyrazolidinyl pyrazolinyl,tetronoyl and tetramoyl groups.

The term “heteroaryl” as employed herein refers to groups having 5 to 14ring atoms; 6, 10 or 14π electrons shared in a cyclic array; andcontaining carbon atoms and 1, 2 or 3 oxygen, nitrogen or sulfurheteroatoms.

Useful heteroaryl groups include thienyl (thiophenyl), benzo[b]thienyl,naphtho[2,3-b]thienyl, thianthrenyl, furyl (furanyl), pyranyl,isobenzofuranyl, chromenyl, xanthenyl, phenoxanthinyl, pyrrolyl,including 1H-pyrrolyl, 2H-pyrrolyl and 3H-pyrrolyl, imidazolyl,pyrazolyl, thiazolyl, isothiazolyl, triazolyl, thiadiazolyl, including1,2,3-thiadiazolyl, pyridyl (pyridinyl), pyrazinyl, pyrimidinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl,purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalzinyl,naphthyridinyl, quinoxalinyl, cinnolinyl, pteridinyl, carbazolyl,β-carbolinyl, phenanthridinyl, acrindinyl, perimidinyl, phenanthrolinyl,phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl,phenoxazinyl, 1,4-dihydroquinoxaline-2,3-dione, 7-aminoisocoumarin,pyrido[1,2-a]pyrimidin-4-one, 1,2-benzoisoxazol-3-yl, benzimidazolyl,2-oxindolyl and 2-oxobenzimidazolyl. Where the heteroaryl group containsa nitrogen atom in a ring, such nitrogen atom may be in the form of anN-oxide, e.g., a pyridyl N-oxide, pyrazinyl N-oxide and pyrimidinylN-oxide.

The term “heteroarylalkyl” is used herein to mean any of theabove-mentioned C₁₋₁₀ alkyl groups substituted by any of theabove-mentioned hetroaryl groups. Both the heteroaryl- and thealkyl-portions of the heteroarylalkyl group may be optionallysubstituted. Typical heteroarylalkyl groups include pyridylmethyl andpyrrolylmethyl.

The term “heteroaryloxy” is used herein to mean oxygen substituted byone of the above-mentioned heteroaryl groups, which may be optionallysubstituted. Useful heteroaryloxy groups include pyridyloxy,pyrazinyloxy, pyrrolyloxy, pyrazolyloxy, imidazolyloxy andthiophenyloxy.

The term “heteroarylalkoxy” is used herein to mean any of the abovementioned C₁₋₁₀ alkoxy groups substituted by any of the above-mentionedheteroaryl groups, which may be optionally substituted.

Some of the compounds of the present invention may exist asstereoisomers, including optical isomers. The invention includes allstereoisomers and both the racemic mixtures of such stereoisomers, aswell as the individual enantiomers that may be separated according tomethods that are well known to those of ordinary skill in the art.

Examples of pharmaceutically acceptable addition salts include inorganicand organic acid addition salts, such as hydrochloride, hydrobromide,phosphate, sulphate, citrate, lactate, tartrate, maleate, fumarate,mandelate and oxalate; and inorganic and organic base addition saltswith bases, such as sodium hydroxy, Tris(hydroxymethyl)aminomethane(TRIS, tromethane) and N-methyl-glucamine.

Examples of prodrugs of the compounds of the invention include thesimple esters of carboxylic acid containing compounds (e.g., thoseobtained by condensation with a C₁₋₄ alcohol according to methods knownin the art); esters of hydroxy containing compounds (e.g., thoseobtained by condensation with a C₁₋₄ carboxylic acid, C₃₋₆ dioic acid oranhydride thereof, such as succinic and fumaric anhydrides according tomethods known in the art); imines of amino containing compounds (e.g.,those obtained by condensation with a C₁₋₄ aldehyde or ketone accordingto methods known in the art); carbamate of amino containing compoundssuch as those described by Leu, et. al., (J. Med. Chem. 42:3623-3628(1999)) and Greenwald, et. al., (J. Med. Chem. 42:3657-3667 (1999)); andacetals and ketals of alcohol containing compounds (e.g., those obtainedby condensation with chloromethyl methyl ether or chloromethyl ethylether according to methods known in the art).

The compounds of this invention may be prepared using methods known tothose skilled in the art, or the novel methods of this invention.Specifically, the compounds of this invention with Formulae I and IIIand V may be prepared as illustrated by the exemplary reaction inScheme 1. Condensation ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide and2-pyridine-carboxaldehyde in the presence of glacial acetic acid inethanol produced the product4-(bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridin-2-ylmethylidene)thiosemicarbazide.

Compounds of this invention with Formulae II, IV and VI may be preparedas illustrated by the exemplary reaction in Scheme 2. ReactionN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide, picolinicacid in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide indichloromethane produced the product4-(bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridine-2-carbonyl)thiosemicarbazide.

An important aspect of the present invention is the discovery thatcompounds having Formulae I-VI are activators of caspases and inducersof apoptosis. Therefore, these compounds are useful in a variety ofclinical conditions in which there is uncontrolled cell growth andspread of abnormal cells, such as in the case of cancer.

Another important aspect of the present invention is the discovery thatcompounds having Formulae I-VI are potent and highly efficaciousactivators of caspases and inducers of apoptosis in drug resistantcancer cells, such as breast and prostate cancer cells, which enablesthese compounds to kill these drug resistant cancer cells. Incomparison, most standard anti-cancer drugs are not effective in killingdrug resistant cancer cells under the same conditions. Therefore,compounds of this invention are useful for the treatment of drugresistant cancer, such as breast cancer in animals.

The present invention includes a therapeutic method useful to modulatein vivo apoptosis or in vivo neoplastic disease, comprisingadministering to a subject in need of such treatment an effective amountof a compound, or a pharmaceutically acceptable salt or prodrug of thecompound of Formulae I-VI, which functions as a caspase cascadeactivator and inducer of apoptosis.

The present invention also includes a therapeutic method comprisingadministering to an animal an effective amount of a compound, or apharmaceutically acceptable salt or prodrug of said compound of FormulaeI-VI, wherein said therapeutic method is useful to treat cancer, whichis a group of diseases characterized by the uncontrolled growth andspread of abnormal cells. Such diseases include, but are not limited to,Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic leukemia,chronic lymphocytic leukemia, multiple myeloma, neuroblastoma, breastcarcinoma, ovarian carcinoma, lung carcinoma, Wilms' tumor, cervicalcarcinoma, testicular carcinoma, soft-tissue sarcoma, primarymacroglobulinemia, bladder carcinomas, chronic granulocytic leukemia,primary brain carcinoma, malignant melanoma, small-cell lung carcinoma,stomach carcinoma, colon carcinoma, malignant pancreatic insulinoma,malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, heador neck carcinoma, osteogenic sarcoma, pancreatic carcinoma, acutegranulocytic leukemia, hairy cell leukemia, neuroblastoma,rhabdomyosarcoma, Kaposi's sarcoma, genitourinary carcinoma, thyroidcarcinomas esophageal carcinoma, malignant hypercalcemia, cervicalhyperplasia, renal cell carcinoma, endometrial carcinoma, polycythemiavera, essential thrombocytosis, adrenal cortex carcinoma, skin cancer,and prostatic carcinoma.

In practicing the therapeutic methods, effective amounts of compositionscontaining therapeutically effective concentrations of the compoundsformulated for oral, intravenous, local and topical application, for thetreatment of neoplastic diseases and other diseases in which caspasecascade mediated physiological responses are implicated, areadministered to an individual exhibiting the symptoms of one or more ofthese disorders. The amounts are effective to ameliorate or eliminateone or more symptoms of the disorders. An effective amount of a compoundfor treating a particular disease is an amount that is sufficient toameliorate, or in some manner reduce, the symptoms associated with thedisease. Such amount may be administered as a single dosage or may beadministered according to a regimen, whereby it is effective. The amountmay cure the disease but, typically, is administered in order toameliorate the symptoms of the disease. Typically, repeatedadministration is required to achieve the desired amelioration ofsymptoms.

In another embodiment, a pharmaceutical composition comprising acompound, or a pharmaceutically acceptable salt of said compound ofFormulae I-VI, which functions as a caspase cascade activator andinducer of apoptosis in combination with a pharmaceutically acceptablevehicle, is provided.

Another embodiment of the present invention is directed to a compositioneffective to inhibit neoplasia comprising a compound, or apharmaceutically acceptable salt or prodrug of said compound of FormulaeI-VI, which functions as a caspase cascade activator and inducer ofapoptosis, in combination with at least one known cancerchemotherapeutic agent, or a pharmaceutically acceptable salt of saidagent. Examples of known cancer chemotherapeutic agents, which may beused for combination therapy include, but not are limited to alkylatingagents, such as busulfan, cis-platin, mitomycin C, and carboplatin;antimitotic agents, such as coichicine, vinblastine, paclitaxel, anddocetaxel; topo I inhibitors, such as camptothecin and topotecan; topoII inhibitors, such as doxorubicin and etoposide; RNA/DNAantimetabolites, such as 5-azacytidine, 5-fluorouracil and methotrexate;DNA antimetabolites, such as 5-fluoro-2′-deoxy-uridine, ara-C,hydroxyurea and thioguanine; and antibodies, such as campath, Herceptin®or Rituxan®. Other known cancer chemotherapeutic agents, which may beused for combination therapy, include melphalan, chlorambucil,cyclophosamide, ifosfamide, vincristine, mitoguazone, epirubicin,aclarubicin, bleomycin, mitoxantrone, elliptinium, fludarabine,octreotide, retinoic acid, tamoxifen, Gleevec® and alanosine.

In practicing the methods of the present invention, the compound of theinvention may be administered together with at least one knownchemotherapeutic agent as part of a unitary pharmaceutical composition.Alternatively, the compound of the invention may be administered apartfrom at least one known cancer chemotherapeutic agent. In oneembodiment, the compound of the invention and at least one known cancerchemotherapeutic agent are administered substantially simultaneously,i.e. the compounds are administered at the same time or one after theother, so long as the compounds reach therapeutic levels in the blood atthe same time. On another embodiment, the compound of the invention andat least one known cancer chemotherapeutic agent are administeredaccording to their individual dose schedule, so long as the compoundsreach therapeutic levels in the blood.

Another embodiment of the present invention is directed to a compositioneffective to inhibit neoplasia comprising a bioconjugates of saidcompound of Formulae I-VI, which functions as a caspase cascadeactivator and inducer of apoptosis, in bioconjugation with at least oneknown therapeutically useful antibody, such as Herceptin® or Rituxan®,growth factors, such as DGF, NGF; cytokines, such as IL-2, IL-4; or anymolecule that binds to the cell surface. The antibodies and othermolecules will deliver the compound of Formulae I-VI to its targets andmake it an effective anticancer agent. The bioconjugates could alsoenhance the anticancer effect of therapeutically useful antibodies, suchas Herceptin® or Rituxan®.

Similarly, another embodiment of the present invention is directed to acomposition effective in inhibiting neoplasia comprising a compound, ora pharmaceutically acceptable salt or prodrug of said compound ofFormulae I-VI, which functions as a caspase cascade activator andinducer of apoptosis, in combination with radiation therapy. In thisembodiment, the compound of the invention may be administered at thesame time as the radiation therapy is administered or at a differenttime.

Yet another embodiment of the present invention is directed to acomposition effective for post-surgical treatment of cancer, comprisinga compound, or a pharmaceutically acceptable salt or prodrug of saidcompound of Formulae I-VI, which functions as a caspase cascadeactivator and inducer of apoptosis. The invention also relates to amethod of treating cancer by surgically removing the cancer and thentreating the animal with one of the pharmaceutical compositionsdescribed herein.

A wide range of immune mechanisms operate rapidly following exposure toan infectious agent. Depending on the type of infection, rapid clonalexpansion of the T and B lymphocytes occurs to combat the infection. Theelimination of the effector cells following an infection is one of themajor mechanisms for maintaining immune homeostasis. The elimination ofthe effector cells has been shown to be regulated by apoptosis.Autoimmune diseases have lately been determined to occur as aconsequence of deregulated cell death. In certain autoimmune diseases,the immune system directs its powerful cytotoxic effector mechanismsagainst specialized cells, such as oligodendrocytes in multiplesclerosis, the beta cells of the pancreas in diabetes mellitus, andthyrocytes in Hashimoto's thyroiditis (Ohsako, S. & Elkon, K. B., CellDeath Differ. 6:13-21 (1999)). Mutations of the gene encoding thelymphocyte apoptosis receptor Fas/APO-1/CD95 are reported to beassociated with defective lymphocyte apoptosis and autoimmunelymphoproliferative syndrome (ALPS), which is characterized by chronic,histologically benign splenomegaly, generalized lymphadenopathy,hypergammaglobulinemia, and autoantibody formation. (Infante, A. J., etal., J. Pediatr. 133:629-633 (1998) and Vaishnaw, A. K., et al., J.Clin. Invest. 103:355-363 (1999)). It was reported that overexpressionof Bcl-2, which is a member of the bcl-2 gene family of programmed celldeath regulators with anti-apoptotic activity, in developing B cells oftransgenic mice, in the presence of T cell dependent costimulatorysignals, results in the generation of a modified B cell repertoire andin the production of pathogenic autoantibodies (Lopez-Hoyos, M., et al.,Int. J. Mol. Med. 1:475-483 (1998)). It is, therefore, evident that manytypes of autoimmune disease are caused by defects of the apoptoticprocess. One treatment strategy for such diseases is to activateapoptosis in the lymphocytes that are causing the autoimmune disease(O'Reilly, L. A. & Strasser, A., Inflamm. Res. 48:5-21 (1999)).

Fas-Fas ligand (FasL) interaction is known to be required for themaintenance of immune homeostasis. Experimental autoimmune thyroiditis(EAT), characterized by autoreactive T and B cell responses and a markedlymphocytic infiltration of the thyroid, is a good model to study thetherapeutic effects of FasL. Batteux, F., et al., (J. Immunol.162:603-608 (1999)) reported that by direct injection of DNA expressionvectors encoding FasL into the inflamed thyroid, the development oflymphocytic infiltration of the thyroid was inhibited and induction ofinfiltrating T cells death was observed. These results show that FasLexpression on thyrocytcs may have a curative effect on ongoing EAT byinducing death of pathogenic autoreactive infiltrating T lymphocytes.

Bisindolylmaleimide VIII is known to potentiate Fas-mediated apoptosisin human astrocytoma 1321N1 cells and in Molt-4T cells; both of whichwere resistant to apoptosis induced by anti-Fas antibody in the absenceof bisindolylmaleimide VIII. Potentiation of Fas-mediated apoptosis bybisindolylmaleimide VIII was reported to be selective for activated,rather than non-activated, T cells, and was Fas-dependent. Zhou T., etal., (Nat. Med. 5:42-48 (1999)) reported that administration ofbisindolylmaleimide VIII to rats during autoantigen stimulationprevented the development of symptoms of T cell-mediated autoimmunediseases in two models, the Lewis rat model of experimental allergicencephalitis and the Lewis adjuvant arthritis model. Therefore, theapplication of a Fas-dependent apoptosis enhancer, such asbisindolylmaleimide VIII, may be therapeutically useful for the moreeffective elimination of detrimental cells and inhibition of Tcell-mediated autoimmune diseases. Therefore, an effective amount of acompound, or a pharmaceutically acceptable salt or prodrug of thecompound of Formulae I-VI, which functions as a caspase cascadeactivator and inducer of apoptosis, would be an effective treatment forautoimmune diseases.

Psoriasis is a chronic skin disease that is characterized by scaly redpatches. Psoralen plus ultraviolet A (PUVA) is a widely used andeffective treatment for psoriasis vulgaris and Coven, et al.,Photodermatol. Photoimmunol. Photomed. 15:22-27 (1999), reported thatlymphocytes treated with psoralen 8-MOP or TMP and UVA, displayed DNAdegradation patterns typical of apoptotic cell death. Ozawa, et al., J.Exp. Med. 189:711-718 (1999) reported that induction of T cell apoptosiscould be the main mechanism by which 312-nm UVB resolves psoriasis skinlesions. Low doses of methotrexate may be used to treat psoriasis torestore a clinically normal skin. Heenen, et al, Arch. Dermatol. Res.290:240-245 (1998), reported that low doses of methotrexate may induceapoptosis and that this mode of action could explain the reduction inepidermal hyperplasia during treatment of psoriasis with methotrexate.Therefore, an effective amount of a compound, or a pharmaceuticallyacceptable salt or prodrug of the compound of Formulae I-VI, whichfunctions as a caspase cascade activator and inducer of apoptosis, wouldbe an effective treatment for hyperproliferative skin diseases, such aspsoriasis.

Synovial cell hyperplasia is a characteristic of patients withrheumatoid arthritis (RA). It is believed that excessive proliferationof RA synovial cells, as well as defects in synovial cell death, may beresponsible for synovial cell hyperplasia. Wakisaka, et al., Clin. Exp.Immunol. 114:119-128 (1998), found that although RA synovial cells coulddie via apoptosis through a Fas/FasL pathway, apoptosis of synovialcells was inhibited by proinflammatory cytokines present within thesynovium. Wakisaka, et al. also suggested that inhibition of apoptosisby the proinflammatory cytokines may contribute to the outgrowth ofsynovial cells, and lead to pannus formation and the destruction ofjoints in patients with RA. Therefore, an effective amount of acompound, or a pharmaceutically acceptable salt or prodrug of thecompound of Formulae I-VI, which functions as a caspase cascadeactivator and inducer of apoptosis, would be an effective treatment forrheumatoid arthritis.

There has been an accumulation of convincing evidence that apoptosisplays a major role in promoting resolution of the acute inflammatoryresponse. Neutrophils are constitutively programmed to undergoapoptosis, thus limiting their pro-inflammatory potential and leading torapid, specific, and non-phlogistic recognition by macrophages andsemi-professional phagocytes (Savill, J., J. Leukoc. Biol. 61:375-380(1997)). Boirivant, et al., Gastroenterology 116:557-565 (1999),reported that lamina propria T cells, isolated from areas ofinflammation in Crohn's disease, ulcerative colitis, and otherinflammatory states, manifest decreased CD2 pathway-induced apoptosis.In addition, studies of cells from inflamed Crohn's disease tissueindicate that this defect is accompanied by elevated Bcl-2 levels.Therefore, an effective amount of a compound, or a pharmaceuticallyacceptable salt or prodrug of the compound of Formulae I-VI, whichfunctions as a caspase cascade activator and inducer of apoptosis, wouldbe an effective treatment for inflammation.

Pharmaceutical compositions within the scope of this invention includeall compositions wherein the compounds of the present invention arecontained in an amount that is effective to achieve its intendedpurpose. While individual needs vary, determination of optimal ranges ofeffective amounts of each component is within the skill of the art.Typically, the compounds may be administered to animals, e.g., mammals,orally at a dose of 0.0025 to 50 mg/kg of body weight, per day, or anequivalent amount of the pharmaceutically acceptable salt thereof, to amammal being treated for apoptosis-mediated disorders. Preferably,approximately 0.01 to approximately 10 mg/kg of body weight is orallyadministered to treat or prevent such disorders. For intramuscularinjection, the dose is generally approximately one-half of the oraldose. For example, a suitable intramuscular dose would be approximately0.0025 to approximately 25 mg/kg of body weight, and most preferably,from approximately 0.01 to approximately 5 mg/kg of body weight. If aknown cancer chemotherapeutic agent is also administered, it isadministered in an amount that is effective to achieve its intendedpurpose. The amounts of such known cancer chemotherapeutic agentseffective for cancer are well known to those of skill in the art.

The unit oral dose may comprise from approximately 0.01 to approximately50 mg, preferably approximately 0.1 to approximately 10 mg of thecompound of the invention. The unit dose may be administered one or moretimes daily as one or more tablets, each containing from approximately0.1 to approximately 10, conveniently approximately 0.25 to 50 mg of thecompound or its solvates.

In a topical formulation, the compound may be present at a concentrationof approximately 0.01 to 100 mg per gram of carrier.

In addition to administering the compound as a raw chemical, thecompounds of the invention may be administered as part of apharmaceutical preparation containing suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries, whichfacilitate processing of the compounds into preparations that may beused pharmaceutically. Preferably, the preparations, particularly thosepreparations which may be administered orally and which may be used forthe preferred type of administration, such as tablets, dragees, andcapsules, and also preparations that may be administered rectally, suchas suppositories, as well as suitable solutions for administration byinjection or orally, that contain from approximately 0.01 to 99 percent,preferably from approximately 0.25 to 75 percent of active compound(s),together with the excipient.

Also included within the scope of the present invention are thenon-toxic pharmaceutically acceptable salts of the compounds of thepresent invention. Acid addition salts are formed by mixing a solutionof the particular apoptosis inducers of the present invention with asolution of a pharmaceutically acceptable non-toxic acid, such ashydrochloric acid, fumaric acid, maleic acid, succinic acid, aceticacid, citric acid, tartaric acid, carbonic acid, phosphoric acid, oxalicacid, and the like. Basic salts are formed by mixing a solution of theparticular apoptosis inducers of the present invention with a solutionof a pharmaceutically acceptable non-toxic base, such as sodiumhydroxide, potassium hydroxide, choline hydroxide, sodium carbonate,Tris, N-methyl-glucamine and the like.

The pharmaceutical compositions of the invention may be administered toany animal which may experience the beneficial effects of the compoundsof the invention. Foremost among such animals are mammals, e.g., humansand veterinary animals, although the invention is not intended to be solimited.

The pharmaceutical compositions of the present invention may beadministered by any means that achieve their intended purpose. Forexample, administration may be by parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal, transdermal, buccal, intrathecal,intracranial, intranasal or topical routes. Alternatively, orconcurrently, administration may be by the oral route. The dosageadministered will be dependent upon the age, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired.

The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral usemay be obtained by combining the active compounds with solid excipients,optionally grinding the resultant mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as saccharides,e.g., lactose or sucrose, mannitol or sorbitol; cellulose preparationsand/or calcium phosphates, e.g. tricalcium phosphate or calcium hydrogenphosphate; as well as binders, such as starch paste using, e.g., maizestarch, wheat starch, rice starch, potato starch, gelatin, tragacanth,methyl cellulose, hydroxypropylmethylcellulose, sodiumcarboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,disintegrating agents may be added, such as the above-mentioned starchesand also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate. Auxiliariesarc, above all, flow-regulating agents and lubricants, e.g., silica,talc, stearic acid or salts thereof, such as magnesium stearate orcalcium stearate, and/or polyethylene glycol. Dragee cores are providedwith suitable coatings which, if desired, are resistant to gastricjuices. For this purpose, concentrated saceharide solutions may be used,which may optionally contain gum arabic, talc, polyvinyl pyrrolidone,polyethylene glycol and/or titanium dioxide, lacquer solutions andsuitable organic solvents or solvent mixtures. In order to producecoatings resistant to gastric juices, solutions of suitable cellulosepreparations such as acetylcellulose phthalate orhydroxypropymethyl-cellulose phthalate, are used. Dye stuffs or pigmentsmay be added to the tablets or dragee coatings, e.g., for identificationor in order to characterize combinations of active compound doses.

Other pharmaceutical preparations which may be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active compounds in the form of granules whichmay be mixed with fillers, such as lactose; binders, such as starches;and/or lubricants, such as talc or magnesium stearate; and, optionally,stabilizers. In soft capsules, the active compounds are preferablydissolved or suspended in suitable liquids, such as fatty oils or liquidparaffin. In addition, stabilizers may be added.

Possible pharmaceutical preparations, which may be used rectallyinclude, e.g., suppositories, which consist of a combination of one ormore of the active compounds with a suppository base. Suitablesuppository bases are, e.g., natural or synthetic triglycerides, orparaffin hydrocarbons. In addition, it is also possible to use gelatinrectal capsules, which consist of a combination of the active compoundswith a base. Possible base materials include, e.g., liquidtriglycerides, polyethylene glycols, or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, e.g.,water-soluble salts and alkaline solutions. In addition, suspensions ofthe active compounds as appropriate oily injection suspensions may beadministered. Suitable lipophilic solvents or vehicles include fattyoils, e.g., sesame oil, or synthetic fatty acid esters, e.g., ethyloleate or triglycerides or polyethylene glycol-400 (the compounds aresoluble in PEG-400), or cremophor, or cyclodextrins. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension include, e.g., sodium carboxymethyl cellulose, sorbitol,and/or dextran. Optionally, the suspension may also contain stabilizers.

In accordance with one aspect of the present invention, compounds of theinvention are employed in topical and parenteral formulations and areused for the treatment of skin cancer.

The topical compositions of this invention are formulated preferably asoils, creams, lotions, ointments and the like by choice of appropriatecarriers. Suitable carriers include vegetable or mineral oils, whitepetrolatum (white soft paraffin), branched chain fats or oils, animalfats and high molecular weight alcohol (greater than C₁₂). The preferredcarriers are those in which the active ingredient is soluble.Emulsifiers, stabilizers, humectants and antioxidants may also beincluded, as well as agents imparting color or fragrance, if desired.Additionally, transdermal penetration enhancers may be employed in thesetopical formulations. Examples of such enhancers are found in U.S. Pat.Nos. 3,989,816 and 4,444,762.

Creams are preferably formulated from a mixture of mineral oil,self-emulsifying beeswax and water in which mixture the activeingredient, dissolved in a small amount of an oil, such as almond oil,is admixed. A typical example of such a cream is one which includesapproximately 40 parts water, approximately 20 parts beeswax,approximately 40 parts mineral oil and approximately 1 part almond oil.

Ointments may be formulated by mixing a solution of the activeingredient in a vegetable oil, such as almond oil with warm softparaffin, and allowing the mixture to cool. A typical example of such anointment is one which includes approximately 30% almond oil andapproximately 70% white soft paraffin by weight.

The following examples are illustrative, but not limiting, of the methodand compositions of the present invention. Other suitable modificationsand adaptations of the variety of conditions and parameters normallyencountered in clinical therapy and which are obvious to those skilledin the art are within the spirit and scope of the invention.

EXAMPLE 14-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(benzylidene)thiosemicarbazide

To a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (100 mg, 0.55mmol) and benzaldehyde (56 μl, 0.55 mmol) in ethanol (5 mL) was added asolution of sodium acetate (45 mg, 0.55 mmol) in water (1 mL) and themixture was stirred at room temperature for 24 h. The mixture wasdiluted with water (1.5 mL) and the resultant precipitate was collectedby filtration and isolated as a white solid (14 mg, 9%). ¹H NMR (CDCl₃):9.84 (s, 1H), 7.88 (s, 1H), 7.64-7.61 (m, 2H), 7.50 (d, J=7.5 Hz, 1H),7.42-7.40 (m, 3H), 6.24-6.14 (m, 2H), 4.314.26 (m, 1H), 3.06 (s, 1H),2.96 (s, 1H), 1.94-1.86 (m, 1H), 1.67 (d, J=9.3 Hz, 1H), 1.52 (d, J=9.0Hz, 1H), 1.46-1.39 (m, 1H).

EXAMPLE 24-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridin-4-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (100 mg, 0.55mmol) and 4-pyridine-carboxaldehyde (54 μL, 0.56 mmol) similar toExample 1 and isolated as a white solid (30 mg, 20%). ¹H NMR (CDCl₃):10.74 (s, 1H), 8.66 (dd, J=1.5, 4.5 Hz, 2H), 7.93 (s, 1H), 7.51 (s, 1H),7.48 (dd, J=1.5, 4.5 Hz, 2H), 6.24-6.13 (m, 2H), 4.29-4.24 (m, 1H), 3.05(s, 1H), 2.97 (s, 1H), 1.94-1.87(m, 1H), 1.68 (d, J=9.0 Hz, 1H), 1.50(d,J=9.0 Hz, 1H), 1.45-1.39 (m, 1H).

EXAMPLE 34-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-methoxybenzylidene)thiosemicarbazide

A mixture of N1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide(100 mg, 0.56 mmol), p-anisaldehyde (69 μL, 0.56 mmol) and glacialacetic acid (31 μl) in ethanol (5 mL) was stirred at room temperaturefor 1 h. The resultant precipitate was collected by filtration andisolated as a white solid (100 mg, 59%). ¹H NMR (CDCl₃): 9.05 (s, 1H),7.72 (s, 1H), 7.57 (d, J=8.7 Hz, 2H), 7.44 (d, J=6.9 Hz, 1H), 6.93 (d,J=8.7 Hz, 2H), 6.23-6.14 (m, 2H), 4.30-4.26 (m, 1H), 3.85 (s, 3H), 3.06(s, 1H), 2.96 (s, 1H), 1.94-1.86 (m, 1H), 1.67 (d, J=9.0 Hz, 1H), 1.51(d, J=9.0 Hz, 1H), 1.45-1.40 (m, 1H).

EXAMPLE 44-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-[4-(trifluoromethyl)benzylidene]thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (100 mg, 0.56mmol) and α,α,α-trifluoro-p-tolualdehyde (76 μL, 0.56 mmol) similar toExample 3 and isolated as a white solid (60 mg, 32%). ¹H NMR (CDCl₃):9.14 (s, 1H), 7.78 (s, 1H), 7.73 (d, J=8.7 Hz, 2H), 7.67 (d, J=8.7 Hz,2H), 7.46 (d, J=7.2 Hz, 1H), 6.25-6.14 (m, 2H), 4.30-4.26 (m, 1H), 3.07(s, 1H), 2.98 (s, 1H), 1.96-1.88 (m, 1H), 1.69 (d, J=9.0 Hz, 1H), 1.51(d, J=8.7 Hz, 1H), 1.46-1.40(m, 1H).

EXAMPLE 54-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-chlorobenzylidene)thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (100 mg, 0.56mmol) and 4-chlorobenzaldehyde (79 mg, 0.56 mmol) similar to Example 3and isolated as a white solid (68 mg, 40%). ¹H NMR (CDCl₃): 9.15 (s,1H), 7.73 (s, 1H), 7.56 (d, J=8.4 Hz, 2H), 7.44 (s, 1H), 7.39 (d, J=8.7Hz, 2H), 6.24-6.14 (m, 2H), 4.30-4.25 (m, 1H), 3.06 (s, 1H), 2.97 (s,1H), 1.95-1.87 (m, 1H), 1.68 (d, J=9.0 Hz, 1H), 1.51 (d, J=9.0 Hz, 1H),1.45-1.39(m, 1H).

EXAMPLE 64-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-nitrobenzylidene)thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (100 mg, 0.56mmol) and 4-nitrobenzaldehyde (85 mg, 0.56 mmol) similar to Example 3and isolated as a yellow solid (84 mg, 47%). ¹H NMR (CDCl₃): 9.80 (s,1H), 8.28 (d, J=9.0 Hz, 2H), 7.90 (s, 1H), 7.78 (d, J=8.7 Hz, 2H), 7.47(d, J=7.2 Hz, 1H), 6.26-6.14 (m, 2H), 4.30-4.26 (m, 1H), 3.08 (s, 1H),2.99 (s, 1H), 1.96-1.89 (m, 1H), 1.70 (d, J=9.0 Hz, 1H), 1.51 (d, J=9.0Hz, 1H), 1.47-1.41 (m, 1H).

EXAMPLE 74-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyrrol-2-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (100 mg, 0.56mmol) and pyrrole-2-carboxaldehyde (53 mg, 0.56 mmol) similar to Example3 and isolated as a yellow oil (3 mg, 2%). ¹H NMR (CDCl₃): 9.28 (s, 1H),8.99 (s, 1H), 7.68 (s, 1H), 7.22 (d, J=7.8 Hz, 1H), 6.94 (s, 1H),6.53-6.51 (m, 1H), 6.29-6.26 (m, 1H), 6.22-6.13 (m, 2H), 4.30-4.24 (m,1H), 3.04 (s, 1H), 2.94 (s, 1H), 1.92-1.84 (m, 1H), 1.65 (d, J=9.0 Hz,1H), 1.50 (d, J=9.0 Hz, 1H), 1.45-1.38 (m, 1H).

EXAMPLE 84-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-hydroxybenzylidene)thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (100 mg, 0.56mmol) and 4-hydroxybenzaldehyde (68 mg, 0.56 mmol) similar to Example 3and isolated as a yellow solid (47 mg, 29%). ¹H NMR (CDCl₃): 9.00 (s,1H), 7.68 (s, 1H), 7.52 (d, J=8.7 Hz, 2H), 7.43 (d, J=6.9 Hz, 1H), 6.88(d, J=8.7 Hz, 2H), 6.23-6.14 (m, 2H), 4.30-4.26 (m, 1H), 3.05 (s, 1H),2.96 (s, 1H), 1.94-1.86 (m, 1H), 1.66 (d, J=9.0 Hz, 1H), 1.51 (d, J=9.0Hz, 1H), 1.45-1.38 (m, 1H).

EXAMPLE 94-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridin-3-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (100 mg, 0.56mmol) and 3-pyridine carboxaldehyde (53 μL, 0.56 mmol) similar toExample 3 and isolated as a white solid (59 mg, 39%). ¹H NMR (CDCl₃):9.51 (s, 1H), 8.85 (d, J=1.8 Hz, 1H), 8.64 (dd, J=1.7, 5.0 Hz, 1H),7.96-7.92 (m, 1H), 7.82 (s, 1H), 7.46 (d, J=6.3 Hz, 1H), 7.38-7.34 (m,1H), 6.24-6.14 (m, 2H), 4.30-4.25 (m, 1H), 3.06 (s, 1H), 2.97 (s, 1H),1.95-1.87 (m, 1H), 1.67 (d, J=8.7 Hz, 1H), 1.5 (d, J=9.0 Hz, 1H),1.45-1.39 (m, 1H).

EXAMPLE 104-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(5-nitrothiophen-2-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (100 mg, 0.56mmol) and 5-nitrothiophene-2-carboxaldehyde (88 mg, 0.56 mmol) similarto Example 3 and isolated as an orange solid (67 mg, 37%). ¹H NMR(CDCl₃): 10.13 (s, 1H), 7.96 (s, 1H), 7.86 (d, J=4.2 Hz, 1H), 7.37 (d,J=7.5 Hz, 1H), 7.17 (d, J=4.2 Hz, 1H), 6.26-6.13 (m, 2H), 4.26-4.20 (m,1H), 3.05 (s, 1H), 3.00 (s, 1H), 1.95-1.87 (m, 1H), 1.70 (d, J=9.0 Hz,1H), 1.49 (d, J=9.0 Hz, 1H), 1.46-1.39 (m, 1H).

EXAMPLE 114-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(5-nitro-furan-2-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (100 mg, 0.56mmol) and 5-nitro-2-furaldehyde (59 μL, 0.56 mmol) similar to Example 3and isolated as a dark yellow solid (4 mg, 2%). ¹H NMR (CDCl₃): 10.02(s, 1H), 7.74 (s, 1H), 7.50 (d, J=7.2 Hz, 1H), 7.39 (d, J=3.9 Hz, 1H),6.88 (d, J=3.9, 1H), 6.26-6.14 (m, 2H), 4.26-4.22 (m, 1H), 3.07 (s, 1H),2.99 (s, 1H), 1.94-1.87 (m, 1H), 1.68 (d, J=9.3 Hz, 1H), 1.51 (d, J=9.0Hz, 1H), 1.48-1.42 (m, 1H).

EXAMPLE 124-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(3-nitrobenzylidene)thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (100 mg, 0.56mmol) and 3-nitrobenzaldehyde (85 mg, 0.56 mmol) similar to Example 3and isolated as a yellow solid (68 mg, 38%). ¹H NMR (CDCl₃): 9.82 (s,1H), 8.48 (t, J=1.8 Hz, 1H), 8.28-8.25 (m, 1H), 7.96 (s, 1H), 7.93 (s,1H), 7.56 (t, J=8.1 Hz, 1H), 7.47 (d, J=7.2 Hz, 1H), 6.26-6.16 (m, 2H),4.32-4.25 (m, 1H), 3.09 (s, 1H), 3.00 (s, 1H), 1.97-1.90 (m, 1H), 1.70(d, J=9.0 Hz, 1H), 1.53 (d, J=9.3 Hz, 1H), 1.49-1.43 (m, 1H).

EXAMPLE 134-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(2-nitrobenzylidene)thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (100 mg, 0.56mmol) and 2-nitrobenzaldehyde (85 mg, 0.56 mmol) similar to Example 3and isolated as a yellow solid (20 mg, 11%). ¹H NMR (CDCl₃): 9.37 (s,1H), 8.27 (s, 1H), 8.00 (d, J=8.1 Hz, 1H), 7.89 (d, J=7.5 Hz, 1H), 7.68(t, J=7.5 Hz, 1H), 7.57 (t, J=7.8 Hz, 1H), 7.43 (d, J=7.2 Hz, 1H),6.24-6.14 (m, 2H), 4.30-4.26 (m, 1H), 3.07 (s, 1H), 2.97 (s, 1H),1.94-1.86 (m, 1H), 1.67 (d, J=8.4 Hz, 1H), 1.52 (d, J=9.0 Hz, 1H),1.46-1.40 (m, 1H).

EXAMPLE 144-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-[3-(trifluoromethyl)benzylidene]thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (100 mg, 0.56mmol) and 3-(trifluoromethyl)benzaldehyde (75 μL, 0.56 mmol) similar toExample 3 and isolated as a white solid (15 mg, 8%). ¹H NMR (CDCl₃):9.43 (s, 1H), 7.86-7.80(m, 3H), 7.67 (d, J=8.1 Hz, 1H), 7.56 (t, J=7.7Hz, 1H), 7.45 (d, J=6.6 Hz, 1H), 6.25-6.15 (m, 2H), 4.32-4.28 (m, 1H),3.08 (s, 1H), 2.99 (s, 1H), 1.96-1.89 (m, 1H), 1.69 (d, J=9.0 Hz, 1H),1.50 (d, J=9.6 Hz, 1H), 1.48-1.41 (m, 1H).

EXAMPLE 154-(Bicyclo[2.2.1]hept-5n-2-yl)-1-[2-(trifluoromethyl)benzylidene]thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (100 mg, 0.56mmol) and 2-(trifluorometbyl)benzaldehyde (74 μL, 0.56 mmol) similar toExample 3 and isolated as a white solid (56 mg, 29%). ¹H NMR (CDCl₃):9.32 (s, 1H), 8.10 (s, 1H), 8.00 (d, J=7.8 Hz, 1H), 7.72 (d, J=7.2 Hz,1H), 7.62 (t, J=7.5 Hz, 1H), 7.52 (t, J=7.5 Hz, 1H), 7.47 (s, 1H),6.24-6.14 (m, 2H), 4.29 (s, 1H), 3.07 (s, 1H), 2.97 (s, 1H), 1.95-1.88(m, 1H), 1.67 (d, J=9.0 Hz, 1H), 1.50 (d, J=9.3 Hz, 1H), 1.45-1.39 (m,1H).

EXAMPLE 164-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridin-2-ylmethylidene)thiosemicarbazide

A mixture of N1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide(1.8 g, 9.82 mmol), 2-pyridinearboxaldehyde (934 μL, 9.82 mmol) andglacial acetic acid (2.5 mL) in ethanol (50 mL) was stirred at roomtemperature for 24 h. The resultant precipitate was collected byfiltration and isolated as a pale white solid (1.9 g, 73%). ¹H NMR(CDCl₃): 9.39 (s, 1H), 8.63 (d, J=4.8 Hz, 1H), 7.88 (s, 1H), 7.82 (d,J=8.1 Hz, 1H), 7.77-7.72 (m, 1H), 7.56 (d, J=7.2 Hz, 1H), 7.33-7.29 (m,1H), 6.23-6.14 (m, 2H), 4.30-4.26 (m, 1H), 3.07 (s, 1H), 2.96 (s, 1H),1.94-1.86 (m, 1H), 1.66 (d, J=8.7 Hz, 1H), 1.52 (d, J=9.0 Hz, 1H),1.47-1.41 (m, 1H).

EXAMPLE 174-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(6-methyl-pyridin-2-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (100 mg, 0.545mmol) and 6-methyl-2-pyridine carboxaldehyde (66 mg, 0.545 mmol) similarto Example 3 and isolated as a pale white solid (83 mg, 53%). ¹H NMR(CDCl₃): 10.46 (s, 1H), 8.00 (s, 1H), 7.68-7.61 (m, 3H), 7.15 (dd,J=2.1, 6.3 Hz, 1H), 6.22-6.13 (m, 2H), 4.28 (s, 1H), 3.07 (s, 1H), 2.94(s, 1H), 1.91-1.83 (m, 1H), 2.57 (s, 3H), 1.64 (d, J=9.0 Hz, 1H), 1.53(d, J=8.7 Hz, 1H), 1.49-1.43 (m, 1H).

EXAMPLE 184-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-[4-cyano-3-(methylthio)-pyridin-2-ylmethylidene]thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (100 mg, 0.545mmol) and 6-formyl-2-(methylthio)nicotinitrile (97 mg, 0.545 mmol)similar to Example 3 and isolated as a yellow solid (100 mg, 53%). ¹HNMR (CDCl₃): 9.68 (s, 1H), 7.87 (s, 1H), 7.81 (d, J=8.4 Hz, 1H), 7.54(d, J=7.8 Hz, 1H), 7.48 (d, J=7.5 Hz, 1H), 6.26-6.14 (m, 2H), 4.29-4.24(m, 1H), 3.07 (s, 1H), 2.99 (s, 1H), 2.66 (s, 3H), 1.97-1.89 (m, 1H),1.69 (d, J=9.0 Hz, 1H), 1.49 (d, J=9.0 Hz, 1H), 1.46-1.39 (m, 1H).

EXAMPLE 19 4-Cyclohexyl-1-(pyridin-2-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture of4-cyclohexyl-3-thiosemicarbazide (100 mg, 0.577 mmol) and2-pyridinecarboxaldehyde (55 μL, 0.577 mmol) similar to Example 3 andisolated as a white solid (40 mg, 26%). ¹H NMR (CDCl₃): 9.16 (s, 1H),8.65-8.63 (m, 1H), 7.87-7.85 (m, 2H), 7.78-7.73 (m, 1H), 7.38 (d, J=7.2Hz, 1H), 7.34-7.27 (m, 1H), 4.344.27 (m, 1H), 2.18-2.12 (m, 2H),1.82-1.75 (m, 2H), 1.50-1.22 (m, 6H).

EXAMPLE 20 4-Benzyl-1-(pyridin-2-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture of4-benzylthiosemicarbazide (100 mg, 0.552 mmol) and2-pyridinecarboxaldehyde (52 μL, 0.552 mmol) similar to Example 3 andisolated as a pale white solid (83 mg, 59%). ¹H NMR (DMSO-d₆): 11.84 (s,1H), 9.27 (t, J=5.7 Hz, 1H), 8.57 (d, J=4.8 Hz, 1H), 8.30 (d, J=8.1 Hz,1H), 8.13 (s, 1H), 7.83 (t, J=7.7 Hz, 1H), 7.40-7.31 (m, 3H), 7.26 (d,J=4.8 Hz, 1H), 4.86 (d, J=6.0 Hz, 2H).

EXAMPLE 21 4-Ethyl-1-(pyridin-2-ylmethylidene)thiosemicarbazide

The title compound was prepared from 4-ethyl-3-thiosemicarbazide (100mg, 0.838 mmol) and 2-pyridinecarboxaldehyde (80 μL, 0.838 mmol) similarto Example 3 and isolated as a white solid (137 mg, 78%). ¹H NMR(DMSO-d₆): 11.65 (s, 1H), 8.72 (t, J=5.4 Hz, 1H), 8.58-8.55 (m, 1H),8.27 (dd, J=1.1, 8.1 Hz, 1H), 8.09 (s, 1H), 7.88-7.82 (m, 1H), 7.40-7.36(m, 1H), 3.65-3.56 (m, 2H), 1.16 (t, J=7.1 Hz, 3H).

EXAMPLE 22 4-(3-Picolyl)-1-(pyridin-2-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture of4-(3-picolyl)-3-thiosemicarbazide (100 mg, 0.549 mmol),2-pyridinecarboxaldehyde (52 μL, 0.549 mmol) and glacial acetic acid (62μL) similar to Example 3 and isolated as a white solid (87 mg, 58%). ¹HNMR (DMSO-d₆): 11.76 (s, 1H), 9.18 (t, J=6.0 Hz, 1H), 8.45-8.43 (m, 2H),8.33 (dd, J=1.7, 4.7 Hz, 1H), 8.15 (d, J=8.4 Hz, 1H), 8.0 (s, 1H), 7.72(t, J=8.0 Hz, 1H), 7.64 (d, J=7.8 Hz, 1H), 7.28-7.22 (m, 2H), 4.74 (d,J=6.3 Hz, 2H).

EXAMPLE 234-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridine-2-carbonyl)thiosemicarbazide

A mixture of N1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide(100 mg, 0.545 mmol), picolinic acid (67 mg, 0.545 mmol) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (104 mg) indichloromethane (5 mL) was stirred at room temperature for 24 h andisolated as a white solid (47 mg, 30%). ¹H NMR (CDCl₃): 10.57 (s, 1H),9.20 (s, 1H), 8.63 (d, J=4.2 Hz, 1H), 8.06 (d, J=7.5 Hz, 1H), 7.89-7.83(m, 1H), 7.51-7.47 (m, 1H), 7.02 (brs, 1H), 6.16-6.06 (m, 2H), 4.11 (s,1H), 2.96 (s, 1H), 2.83 (s, 1H), 1.84-1.76 (m, 1H), 1.38-1.28 (m, 3H).

EXAMPLE 24 4-(2,4-Dimethylphenyl)-1-(benzylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (100 mg, 0.512 mmol) andbenzaldehyde (52 μL, 0.512 mmol) similar to Example 3 and isolated as awhite solid (110 mg, 76%). ¹H NMR (CDCl₃): 9.43 (s, 1H), 8.90 (s, 1H),7.86 (s, 1H), 7.69-7.66 (m, 2H), 7.50 (d, J=7.5 Hz, 1H), 7.44-7.42 (m,3H), 7.09 (d, J=8.1 Hz, 2H), 2.35 (s, 3H), 2.32 (s, 3H).

EXAMPLE 254-(2,4-Dimethylphenyl)-1-[4-(trifluoromethyl)benzylidene]thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (100 mg, 0.512 mmol) and4-(trifluoromethyl)benzaldehyde (70 μL, 0.512 mmol) similar to Example 3and isolated as a white solid (123 mg, 65%). ¹H NMR (CDCl₃): 9.69 (s,1H), 8.88 (s, 1H), 7.91 (s, 1H), 7.79 (d, J=7.5 Hz, 2H), 7.68 (d, J=7.8Hz, 2H), 7.48 (d, J=7.5 Hz, 1H), 7.10 (d, J=9.3 Hz, 2H), 2.36 (s, 3H),2.32 (s, 3H).

EXAMPLE 264-(2,4-Dimethylphenyl)-1-(4-cyanobenzylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (100 mg, 0.512 mmol) and4-cyanobenzaldehyde (67 mg, 0.512 mmol) similar to Example 3 andisolated as a white solid (100 mg, 60%). ¹H NMR (CDCl₃): 9.61 (s, 1H),8.84 (s, 1H), 7.86 (s, 1H), 7.78 (d, J=8.4 Hz, 2H), 7.71 (d, J=8.7 Hz,2H), 7.45 (d, J=8.1 Hz, 1H), 7.10 (d, J=10.8 Hz, 2H), 2.36 (s, 3H), 2.31(s, 3H).

EXAMPLE 274-(2,4-Dimethylphenyl)-1-(4-methoxybenzylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (100 mg, 0.512 mmol) andp-anisaldehyde (62 μL, 0.512 mmol) similar to Example 3 and isolated asa white solid (109 mg, 64%). ¹H NMR (CDCl₃): 9.11 (s, 1H), 8.86 (s, 1H),7.78 (s, 1H), 7.62 (d, J=8.7 Hz, 2H), 7.51 (d, J=7.8 Hz, 1H), 7.08 (d,J=8.4 Hz, 2H), 6.95 (d, J=8.7 Hz, 2H), 3.86 (s, 3H), 2.35 (s, 3H), 2.32(s, 3H).

EXAMPLE 284-(2,4-Dimethylphenyl)-1-(4-chlorobenzylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (100 mg, 0.512 mmol) and4-chlorobenzaldehyde (72 mg, 0.512 mmol) similar to Example 3 andisolated as a white solid (121 mg, 70%). ¹H NMR (CDCl₃): 9.46 (s, 1H),8.84 (s, 1H), 7.82 (s, 1H), 7.61 (d, J=8.7 Hz, 2H), 7.47 (d, J=8.1 Hz,1H), 7.40 (d, J=8.7 Hz, 2H), 7.09 (d, J=8.4 Hz, 2H), 2.35 (s, 3H), 2.31(s, 3H).

EXAMPLE 294-(2,4-Dimethylphenyl)-1-(6-methyl-pyridin-2-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (100 mg, 0.512 mmol) and6-methyl-2-pyridinecarboxaldehyde (62 mg, 0.512 mmol) similar to Example3 and isolated as a white solid (107 mg, 66%). ¹H NMR (CDCl₃): 9.21 (s,1H), 8.96 (s, 1H), 7.88 (s, 1H), 7.72-7.62 (m, 2H), 7.47 (d, J=8.1 Hz,1H), 7.19 (d, J=7.5 Hz, 1H), 7.09 (d, J=8.4 Hz, 2H), 2.59 (s, 3H), 2.35(s, 3H), 2.32 (s, 3H).

EXAMPLE 304-(2,4-Dimethylphenyl)-2-(4-nitrobenzylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (100 mg, 0.512 mmol) and4-nitrobenzaldehyde (77 mg, 0.512 mmol) similar to Example 3 andisolated as a yellow solid (95 mg, 54%). ¹H NMR (DMSO-d₆): 12.06 (brs,1H), 10.14 (s, 1H), 8.24 (d, J=9.0 Hz, 2H), 8.20 (s, 1H), 8.18 (d, J=9.0Hz, 2H), 7.11 (d, J=8.1 Hz, 1H), 7.04 (d, J=8.1 Hz, 2H), 2.31 (s, 3H),2.18 (s, 3H).

EXAMPLE 314-(2,4-Dimethylphenyl)-1-(pyridin-3-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (100 mg, 0.512 mmol) and3-pyridinecarboxaldehyde (49 μL, 0.512 mmol) similar to Example 3 andisolated as a white solid (94 mg, 61%). ¹H NMR (CDCl₃): 9.96 (s, 1H),8.87 (d, J=1.8 Hz, 2H), 8.65 (dd, J=1.8, 5.1 Hz, 1H), 8.03-7.99 (m, 1H),7.93 (s, 1H), 7.47 (d, J=8.1 Hz, 1H), 7.39-7.35 (m, 1H), 7.10 (d, J=8.7Hz, 2H).

EXAMPLE 324-(2,4-Dimethylphenyl)-1-[4-cyano-3-(methylthio)-pyridin-2-ylmethylidene]thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (100 mg, 0.512 mmol) and6-formyl-2-(methylthio)nicotinonitrile (91 mg, 0.512 mmol) similar toExample 3 and isolated as a yellow solid (138 mg, 72%). ¹H NMR(DMSO-d₆): 12.23 (s, 1H), 10.25 (s, 1H), 8.37 (d, J=7.8 Hz, 1H), 8.24(d, J=8.1 Hz, 1H), 8.12 (s, 1H), 7.11-7.05 (m, 3H), 2.64 (s, 3H), 2.30(s, 3H), 2.17 (s, 3H).

EXAMPLE 334-(2,4-Dimethylphenyl)-1-(pyridin-4-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (100 mg, 0.512 mmol) and4-pyridinecarboxaldehyde (49 μl, 0.512 mmol) similar to Example 3 andisolated as a white solid (105 mg, 68%). ¹H NMR (CDCl₃): 9.86 (s, 1H),8.88 (s, 1H), 8.69 (d, J=6.0 Hz, 2H), 7.83 (s, 1H), 7.53 (d, J=6.0 Hz,2H), 7.46 (d, J=7.8 Hz, 1H), 7.10 (d, J=9.9 Hz, 2H), 2.36 (s, 3H), 2.32(s, 3H).

EXAMPLE 344-(2,4-Dimethylphenyl)-2-(4-hydroxybenzylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (100 mg, 0.512 mmol) and4-hydroxybenzaldehyde (63 mg, 0.512 mmol) similar to Example 3 andisolated as a yellow solid (81 mg, 53%). ¹H NMR (DMSO-d₆): 11.57 (s,1H), 9.75 (s, 1H), 8.03 (s, 1H), 7.70 (d, J=8.4 Hz, 2H), 7.14 (d, J=7.8Hz, 1H), 7.07-7.00 (m, 2H), 6.78 (d, J=8.7 Hz, 2H), 3.17 (s, 1H), 2.29(s, 3H), 2.18 (s, 3H).

EXAMPLE 354-(2,4-Dimethylphenyl)-1-(cyclohexyl-methylidene)thiosemicarbazide

The tide compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) andcyclohexane-carboxaldehyde (31 μL, 0.256 mmol) similar to Example 3 andisolated as a white solid (65 mg, 88%). ¹H NMR (CDCl₃): 9.07 (s, 1H),8.74 (s, 1H), 7.48 (d, J=7.8 Hz, 1H), 7.13 (dd, J=0.9, 5.4 Hz, 1H), 7.07(s, 1H), 7.04 (s, 1H), 2.33 (s, 3H), 2.27 (s, 3H), 1.87-1.69 (m, 5H),1.35-1.24 (m, 5H).

EXAMPLE 364-(2,4-Dimethylphenyl)-1-(3-carboxybenzylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and3-carboxybenzaldehyde (38 mg, 0.256 mmol) similar to Example 3 andisolated as a white solid (64 mg, 76%). ¹H NMR (DMSO-d₆): 11.79 (s, 1H),10.00 (s, 1H), 8.28 (s, 1H), 8.22 (d, J=8.4 Hz, 1H), 8.19 (s, 1H), 7.95(d, J=7.8 Hz, 1H), 7.53 (t, J=7.8 Hz, 1H), 7.12 (d, J=8.1 Hz, 1H), 7.08(s, 1H), 7.02 (d, J=7.8 Hz, 1H), 2.30 (s, 3H), 2.19 (s, 3H).

EXAMPLE 374-(2,4-Dimethylphenyl)-1-(2-carboxybenzylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and2-carboxybenzaldehyde (38 mg, 0.256 mmol) similar to Example 3 andisolated as a white solid (77 mg, 92%). ¹H NMR (DMSO-d₆): 11.91 (s, 1H),9.89 (s, 1H), 8.88 (s, 1H), 8.42 (d, J=8.1 Hz, 1H), 7.85 (dd, J=1.8, 8.1Hz, 1H), 7.59-7.54 (m, 1H), 7.51-7.45 (m, 1H), 7.14 (d, J=8.1 Hz, 1H),7.08 (s, 1H), 7.02 (d, J=7.8 Hz, 1H), 5.76 (s, 1H), 2.29 (s, 3H), 2.18(s, 3H).

EXAMPLE 384-(2,4-Dimethylphenyl)-1-(4-carboxybenzylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and4-carboxybenzaldehyde (38 mg, 0.256 mmol) similar to Example 3 andisolated as a white solid (75 mg, 89%). ¹H NMR (DMSO-d₆): 11.89 (s, 1H),10.02 (s, 1H), 8.17 (s, 1H), 8.01 (d, J=8.7 Hz, 2H), 7.94 (d, J=8.1 Hz,2H), 7.11 (d, J=8.1 Hz, 1H), 7.09 (s, 1H), 7.02 (d, J=8.1 Hz, 1H), 5.75(s, 1H), 2.30 (s, 3H), 2.18 (s, 3H).

EXAMPLE 394-(2,4-Dimethylphenyl)-1-(3-nitrobenzylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and3-nitrobenzaIdehyde (39 mg, 0.256 mmol) similar to Example 3 andisolated as a pale white solid (50 mg, 60%). ¹H NMR (DMSO-d₆): 11.94 (s,1H), 10.14 (s, 1H), 8.76 (s, 1H), 8.31 (d, J=7.8 Hz, 1H), 8.23 (s, 1H),8.20 (dd, J=1.2, 2.4 Hz, 1H), 7.70 (t, J=8.0 Hz, 1H), 7.12 (d, J=8.1 Hz,1H), 7.10 (s, 1H), 7.03 (d, J=8.1 Hz, 1H), 2.30 (s, 3H), 2.19 (s, 3H).

EXAMPLE 404-(2,4-Dimethylphenyl)-1-(2-nitrobenzylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and2-nitrobenzaldehyde (39 mg, 0.256 mmol) similar to Example 3 andisolated as a yellow solid (24 mg, 29%). ¹H NMR (DMSO-d₆): 12.05 (s,1H), 10.02 (s, 1H), 8.59 (d, J=7.8 Hz, 1H), 8.55 (s, 1H), 8.04 (dd,J=1.4, 8.0 Hz, 1H), 7.74 (t, J=7.2 Hz, 1H), 7.66-7.60 (m, 1H), 7.12 (d,J=7.8 Hz, 1H), 7.09 (s, 1H), 7.03 (d, J=8.1 Hz, 1H), 2.30 (s, 3H), 2.18(s, 3H).

EXAMPLE 414-(2,4-Dimethylphenyl)-1-(3-chlorobenzylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and3-chlorobenzaldehyde (29 μL, 0.256 mmol) similar to Example 3 andisolated as a white solid (78 mg, 96%). ¹H NMR (DMSO-d₆): 11.84 (s, 1H),10.04 (s, 1H), 8.20 (s, 1H), 8.09 (s, 1H), 7.72-7.68 (m, 1H), 7.45-7.43(m, 2H), 7.10 (d, J=7.8 Hz, 1H), 7.09 (s, 1H), 7.03 (d, J=7.8 Hz, 1H),2.30 (s, 3H), 2.18 (s, 3H).

EXAMPLE 424-(2,4-Dimethylphenyl)-1-(2-chlorobenzylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and2-chlorobenzaldehyde (29 μL, 0.256 mmol) similar to Example 3 andisolated as a white solid (76 mg, 93%). ¹H NMR (DMSO-d₆): 11.94 (s, 1H),10.01 (s, 1H), 8.56 (s, 1H), 8.46 (d, J=7.8 Hz, 1H), 7.50 (dd, J=1.7,8.0 Hz, 1H), 7.44-7.33 (m, 2H), 7.12 (d, J=7.8 Hz, 1H), 7.09 (s, 1H),7.03 (d, J=8.1 Hz, 1H), 2.30 (s, 3H), 2.18 (s, 3H).

EXAMPLE 434-(2,4-Dimethylphenyl)-1-(3-hydroxybenzylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and3-hydroxybenzaldehyde (31 mg, 0.256 mmol) similar to Example 3 andisolated as a white solid (63 mg, 82%). ¹H NMR (DMSO-d₆): 11.69 (s, 1H),9.85 (s, 1H), 9.56 (s, 1H), 8.04 (s, 1H), 7.29-7.17 (m, 3H), 7.14 (d,J=7.8 Hz, 1H), 7.07 (s, 1H), 7.01 (d, J=8.4 Hz, 1H), 6.81 (d, J=8.4 Hz,1H), 2.29 (s, 3H), 2.18 (s, 3H).

EXAMPLE 444-(2,4-Dimethylphenyl)-1-(2-hydroxybenzylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and2-hydroxybenzaldehyde (27 μL, 0.256 mmol) similar to Example 3 andisolated as a white solid (61 mg, 80%). ¹H NMR (DMSO-d₆): 11.70 (s, 1H),9.94 (s, 1H), 9.83 (s, 1H), 8.45 (s, 1H), 8.07 (d, J=8.1 Hz, 1H),7.24-7.19 (m, 1H), 7.14 (d, J=7.8 Hz, 1H), 7.07 (s, 1H), 7.01 (d, J=7.8Hz, 1H), 6.87 (dd, J=0.9, 8.1 Hz, 1H), 6.81 (t, J=7.7 Hz, 1H), 2.29 (s,3H), 2.18 (s, 3H).

EXAMPLE 454-(2,4-Dimethylphenyl)-1-(4-dimethylaminobenzylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and4-dimethylaminobenzaldehyde (38 mg, 0.256 mmol) similar to Example 3 andisolated as a white solid (78 mg, 93%). ¹H NMR (DMSO-d₆): 11.49 (s, 1H),9.67 (s, 1H), 8.01 (s, 1H), 7.66 (d, J=8.7 Hz, 2H), 7.19 (d, J=7.8 Hz,1H), 7.07 (s, 1H), 7.01 (d, J=8.7 Hz, 1H), 6.71 (d, J=8.7 Hz, 2H), 2.97(s, 6H), 2.29 (s, 3H), 2.19 (s, 3H).

EXAMPLE 464-(2,4-Dimethylphenyl)-1-(furan-2-ylmethylidine)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and furfural (21μL, 0.256 mmol) similar to Example 3 and isolated as a pale white solid(58 mg, 83%). ¹H NMR (CDCl₃): 9.56 (s, 1H), 8.86 (s, 1H), 7.72 (s, 1H),7.54 (d, J=1.8 Hz, 1H), 7.42 (d, J=7.8 Hz, 1H), 7.09 (s, 1H), 7.06 (s,1H), 6.76 (d, J=3.3 Hz, 1H), 6.53-6.51 (m, 1H), 2.34 (s, 3H), 2.30 (s,3H).

EXAMPLE 474-(2,4-Dimethylphenyl)-1-(thiophen-2-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and2-thiophenecarboxaldehyde (36 μL, 0.256 mmol) similar to Example 3 andisolated as a white solid (60 mg, 80%). ¹H NMR (DMSO-d₆): 11.75 (s, 1H),9.56 (s, 1H), 8.32 (s, 1H), 7.68 (dd, J=0.6, 5.1 Hz, 1H), 7.52 (d, J=3.6Hz, 1H), 7.27 (d, J=8.1 Hz, 1H), 7.15-7.12 (m, 1H), 7.07 (s, 1H), 7.01(d, J=7.8 Hz, 1H), 2.29 (s, 3H), 2.19 (s, 3H).

EXAMPLE 484-(2,4-Dimethylphenyl)-1-(5-nitrofuran-2-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and5-nitrofurylaldehyde (36 μL, 0.31 mmol) similar to Example 3 andisolated as a yellow solid (65 mg, 80%). ¹H NMR (DMSO-d₆): 9.93 (s, 1H),8.91 (s, 1H), 7.74 (s, 1H), 7.41 (d, J=7.5 Hz, 1H), 7.39 (d, J=4.0 Hz,1H), 7.07-7.14 (m, 3H), 6.90 (d, J=4.0 Hz, 1H), 2.35 (s, 3H), 2.31 (s,3H).

EXAMPLE 494-(2,4-Dimethylphenyl)-1-(4,5-dimethylfuran-2-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and4,5-dimethylfurylaldehyde (35 μL, 0.31 mmol) similar to Example 3 andisolated as an off-white solid (45 mg, 58%). ¹H NMR (DMSO-d₆): 9.43 (s,1H), 8.84 (s, 1H), 7.60 (s, 1H), 7.40 (d, J=7.8 Hz, 1H), 7.24-7.05 (m,2H), 6.50 (s 1H), 2.34 (s, 3H), 2.30 (s, 3H), 2.27 (s, 3H), 1.96 (s,3H).

EXAMPLE 504-(2,4-Dimethylphenyl)-1-(N-methylpyrrol-2-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) andN-methyl-2-carboaldehydepyrrole (37 μL, 0.31 mmol) similar to Example 3and isolated as an off-white solid (60 mg, 82%). ¹H NMR (DMSO-d₆): 9.33(s, 1H), 8.47 (s, 1H), 7.68 (s, 1H), 7.45 (d, J=8.8 Hz, 1H), 6.97-6.56(m, 2H), 6.44 (dd, J=4.7, 2.1, 1H), 6.06 (dd, J=4.7, 2.1, 1H), 3.76 (s,3H), 2.22 (s, 3H), 2.18 (s, 3H).

EXAMPLE 514-(2,4-Dimethylphenyl)-1-(5-nitrothiophen-2-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and5-nitro-2-thiophenecarboxyaldehyde (32 μL, 0.31 mmol) similar to Example3 and isolated as a yellow solid (50 mg, 58%). ¹H NMR (DMSO-d₆): 9.37(s, 1H), 8.25 (s, 1H), 7.81 (d, J=4.4 Hz, 1H), 7.36 (d, J=7.6 Hz, 1H),7.27 (d, J=4.4 Hz, 1H), 7.08-7.02 (m, 2H), 2.34 (s, 3H), 2.28 (s, 3H).

EXAMPLE 524-(2,4-Dimethylphenyl)-1-(imidazol-2-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and2-formylimidazole (39 mg, 0.31 mmol) similar to Example 3 and isolatedas an off-white solid (17 mg, 24%). ¹H NMR (DMSO-d₆): 9.64 (s, 1H), 7.83(s, 1H), 7.54 (s, 1H), 7.00 (d, J=7.8 Hz, 1H), 6.87-6.82 (m, 3H), 2.15(s, 3H), 2.09 (s, 3H).

EXAMPLE 534-(2,4-Dimethylphenyl)-1-(2-methoxy-4-dimethylaminobenzylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and2-methoxy-4-dimethylaminophenylaldehyde (55 mg, 0.31 mmol) similar toExample 3 and isolated as an off-white solid (34 mg, 39%). ¹H NMR(DMSO-d₆): 8.90 (s, 1H), 8.12 (s, 1H), 7.72 (d, J=8.7 Hz, 1H), 7.53 (d,J=8.1 Hz, 1H), 7.25 (s, 1H), 7.07-7.04 (m, 2H), 6.33 (dd, J=8.7, 2.1,1H), 6.12 (d, J=2.1 Hz, 1H), 3.04 (s, 6H), 2.33 (s, 3H), 2.30 (s, 3H).

EXAMPLE 544-(2,4-Dimethylphenyl)-1-(6-bromo-pyridin-2-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and6-bromo-2-pyridinylcarboxyaldehyde (59 mg, 0.31 mmol) similar to Example3 and isolated as a yellow solid (34 mg, 38%). ¹H NMR (DMSO-d₆): 12.03(s, 1H), 9.81 (s, 1H), 8.35 (s, 1H), 8.11 (s, 1H), 7.61 (t, J=8.1 Hz,1H), 7.49 (d, J=8.0 Hz, 1H), 7.23 (d, J=7.5 Hz, 1H), 7.07-7.01 (m, 2H),2.33 (s, 3H), 2.25 (s, 3H).

EXAMPLE 554-(2,4-Dimethylphenyl)-1-(imidazol-4-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and4-imidazolcarboxyaldehyde (39 mg, 0.31 mmol) similar to Example 3 andisolated as an off-white solid (40 mg, 57%). ¹H NMR (DMSO-d₆): 11.95 (s,1H), 9.91 (s, 1H), 8.35 (s, 1H), 7.98 (s, 1H), 7.90 (s, 1H), 7.69 (d,J=8.0 Hz, 1H), 7.33 (s, 1H), 7.15-7.01 (m, 3H), 2.33 (s, 3H), 2.25 (s,3H).

EXAMPLE 564-(2,4-Dimethylphenyl)-1-(4-morpholino-3-nitrobenzylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and4-morpholino-3-nitrobonzaldehyde (72 mg, 0.31 mmol) similar to Example 3and isolated as an off-white solid (34 mg, 33%). ¹H NMR (DMSO-d₆): 10.06(s, 1H), 8.82 (s, 1H), 8.10 (d, J=2.1 Hz, 1H), 7.86 (s, 1H), 7.72 (dd,J=8.4, 2.1 Hz, 1H), 7.46 (d, J=8.1 Hz, 1H), 7.26-7.0 7 (m, 3H), 3.85 (m,4H), 3.13 (m, 4H), 2.35 (s, 3H), 2.32 (s, 3H).

EXAMPLE 574-(2,4-Dimethylphenyl)-1-(4-bromo-1H-pyrazol-5-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and4-bromo-1H-pyrazole-5-carboxaldehyde (54 mg, 0.31 mmol) similar toExample 3 and isolated as an off-white solid (67 mg, 74%). ¹H NMR(DMSO-d₆): 9.98 (s, 1H), 8.15 (s, 1H), 8.02 (s, 1H), 7.86 (s, 1H),7.16-7.0 3 (m, 3H), 3.85 (m, 4H), 3.13 (m, 4H), 2.30 (s, 3H), 2.18 (s,3H).

EXAMPLE 584-(2,4-Dimethylphenyl)-1-(4-chloro-1-methylpyrazol-3-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and4-chloro-1-methylpyrazole-3-carboxaldehyde (44 mg, 0.31 mmol) similar toExample 3 and isolated as a light yellow solid (54 mg, 66%). ¹H NMR(DMSO-d₆): 9.03 (s, 1H), 7.86 (s, 1H), 7.25 (d, J=7.5 Hz, 1H), 7.23 (s,1H), 7.20 (s, 1H), 7.02 (m, 1H), 3.84 (s, 3H), 2.35 (s, 3H), 2.32 (s,3H).

EXAMPLE 594-(2,4-Dimethylphenyl)-1-(1,2,3-thiazol-4-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and1,2,3-thiazole-4-carboxaldehyde (23 mg, 0.31 mmol) similar to Example 3and isolated as an off-white solid (38 mg, 51%). ¹H NMR (DMSO-d₆): 9.06(s, 1H), 8.47 (s, 1H), 7.38 (s, 1H), 7.29 (d, J=7.8 Hz, 1H), 7.07 (m,2H), 3.84 (s, 3H), 2.35 (s, 3H), 2.29 (s, 3H).

EXAMPLE 604-(2,4-Dimethylphenyl)-1-(thiazol-2-ylethylidene)thiosemicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylthiosemicarbazide (50 mg, 0.256 mmol) and2-acetylthiazole (30 mg, 0.31 mmol) similar to Example 3 and isolated asan off white solid (30 mg, 38%). ¹H NMR (DMSO-d₆): 10.05 (s, 1H), 9.62(s, 1H), 7.88 (d, J=4.5 Hz, 1H), 7.76 (d, J=4.5 Hz, 1H), 7.34 (d, J=7.5Hz, 1H), 7.23-7.06 (m, 2H), 2.24 (s, 3H), 2.29 (s, 3H), 2.21 (s, 3H).

EXAMPLE 614-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(thiazol-2-ylethylidene)thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (50 mg, 0.273mmol) and 2-acetylthiazole (41 mg, 0.33 mmol) similar to Example 3 andisolated as an off-white solid (30 mg, 37%). ¹H NMR (DMSO-d₆): 9.98 (s,1H), 9.62 (s, 1H), 7.89 (m, 2H), 7.77 (m, 1H), 6.16 (ddd, J=8.1, 5.1,2.7 Hz, 2H), 4.23 (m, 1H), 3.04 (s, 2H), 2.23 (s, 3H), 1.87-1.50 (m,4H).

EXAMPLE 624-(2,4-Dimethylphenyl)-1-(2-furan-2-ylmethylidene)semicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylsemicarbazide (55 mg, 0.307 mmol) and2-furylcarboxaldehyde (35 mg, 0.367 mmol) similar to Example 3 andisolated as a white solid (65 mg, 82%). ¹H NMR (DMSO-d₆): 9.81 (s, 1H),7.99 (s, 1H), 7.72 (s, 1H), 7.70 (d, J=2.7 Hz, 1H), 7.50 (d, J=2.4 Hz,1H), 7.05 (d, J=2.4 Hz, 1H), 7.03 (s, 1H), 6.64 (d, J=6.6 Hz, 1H), 6.47(dd, J=1.8, 6.6 Hz, 1H), 2.31 (s, 6H).

EXAMPLE 63 4-(2,4-Dimethylphenyl)-1-(3-chlorobenzylidene)semicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylsemicarbazide (50 mg 0.279 mmol) and3-chlorobenzaldehyde (47 mg, 0.335 mmol) similar to Example 3 andisolated as a white solid (60 mg, 71%). ¹H NMR (DMSO-d₆): 10.85 (s, 1H),8.60 (s, 1H), 7.99 (s, 1H), 7.90 (s, 1H), 7.46-7.35 (m, 3H), 7.04-6.91(m, 2H), 2.62 (s, 3H), 2.23 (s, 3H).

EXAMPLE 64 4-(2,4-Dimethylphenyl)-1-(benzylidene)semicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylsemicarbazide (55 mg 0.279 mmol) and benzaldehyde (35mg, 0.335 mmol) similar to Example 3 and isolated as a white solid (50mg, 67%). ¹H NMR (DMSO-d₆): 10.85 (s, 1H), 8.50 (s, 1H), 7.99 (s, 1H),7.98 (s, 1H), 7.94 (s, 1H), 7.88 (brs, 1H), 7.50-7.35 (m, 3H), 7.04-6.95(m, 2H), 2.62 (s, 3H), 2.23 (s, 3H).

EXAMPLE 654-(2,4-Dimethylphenyl)-1-(4-pyrrolidinobenzylidene)semicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylsemicarbazide (100 mg 0.559 mmol) and4-pyrrolidinobenzaldehyde (117 mg, 0.67 mmol) similar to Example 3 andisolated as an off-white solid (90 mg, 48%). ¹H NMR (DMSO-d₆): 10.25 (s,1H), 8.37 (brs, 1H), 7.89 (s, 1H), 7.40-7.62 (m, 3H), 7.08-6.88 (m, 2H),6.62 (brs, 2H), 3.23 (m, 4H), 2.64 (s, 3H), 2.60 (s, 3H), 2.12 (m, 4H).

EXAMPLE 664-(2,4-Dimethylphenyl)-1-(pyridin-2-ylmethylidene)semicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylsemicarbazide (100 mg 0.559 mmol) and2-pyridylcarboxaldehyde (71 mg, 0.67 mmol) similar to Example 3 andisolated as a light yellow solid (30 mg, 20%). ¹H NMR (DMSO-d₆): 10.78(s, 1H), 8.59 (brs, 1H), 8.24 (brs, 1H), 7.79 (s, 1H), 7.68-6.75 (m,2H), 7.36-7.28 (m, 2H), 6.88-7.0 (m, 2H), 2.30 (s, 3H), 2.17 (s, 3H).

EXAMPLE 674-(2,4-Dimethylphenyl)-1-(2-methoxy-4-dimethylaminobenzylidene)semicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylsemicarbazide (40 mg 0.223 mmol) and2-methoxy-4-dimethylaminobenzaldehyde (50 mg, 0.268 mmol) similar toExample 3 and isolated as a white solid (30 mg, 39%). ¹H NMR (DMSO-d₆):10.35 (s, 1H), 8.30 (s, 1H), 8.18 (s, 1H), 7.77 (d, J=8.8 Hz, 1H), 7.64(d, J=8.8 Hz, 1H), 7.01-6.95 (m, 2H), 6.37 (dd, J=8.8, 2.7 Hz, 1H), 6.25(d, J=2.7 Hz, 1H), 3.83 (s, 3H), 3.29 (s, 6H), 2.24 (s, 3H), 2.22 (s,3H).

EXAMPLE 684-(2,4-Dimethylphenyl)-1-(4-chloro-1-methylpyrazol-3-ylmethylidene)semicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylsemicarbazide (40 mg 0.223 mmol) and4-chloro-1-methylpyrazole-3-carboxyaldehyde (30 mg, 0.268 mmol) similarto Example 3 and isolated as a white solid (30 mg, 49%). ¹H NMR(DMSO-d₆): 10.95 (s, 1H), 8.23 (s, 1H), 8.08 (s, 1H), 7.98 (s, 1H), 7.86(d, J=7.6 Hz, 1H), 7.05-6.97 (m, 3H), 3.83 (s, 3H), 2.25 (s, 3H), 2.23(s, 3H).

EXAMPLE 69 4-(2,4-Dimethylphenyl)-1-(3-hydroxybenzylidene)semicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylsemicarbazide (40 mg 0.223 mmol) and3-hydroxybenzaldehyde (33 mg, 0.268 mmol) similar to Example 3 andisolated as a white solid (30 mg, 47%). ¹H NMR (DMSO-d₆): 10.56 (s, 1H),9.78 (s, 1H), 8.37 (s, 1H), 7.84 (s, 1H), 7.57 (d, J=7.8 Hz, 1H),7.18-7.22 (m, 2H), 7.11-6.98 (m, 2H), 6.79 (brs, 1H), 2.30 (s, 3H), 2.17(s, 3H).

EXAMPLE 704-(2,4-Dimethylphenyl)-1-(2-hydroxy-4-methoxybenzylidene)semicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylsemicarbazide (40 mg, 0.223 mmol) and2-hydroxy-4-methoxybenzaldehyde (40 mg, 0.268 mmol) similar to Example 3and isolated as a white solid (30 mg, 43%). ¹H NMR (DMSO-d₆): 12.23 (s,1H), 10.78 (s, 1H), 8.36 (s, 1H), 8.21 (s, 1H), 7.63 (d, J=7.8 Hz, 1H),7.58 (d, J=8.1 Hz, 1H), 7.12-6.95 (m, 2H), 6.42-6.38 (m, 2H), 3.83 (s,3H), 2.30 (s, 3H), 2.17 (s, 3H).

EXAMPLE 714-(2,4-Dimethylphenyl)-1-(1-methylpyrrol-2-ylmethylidene)semicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylsemicarbazide (40 mg, 0.223 mmol) and1-methyl-2-pyrrolecarboxaldehyde (29 mg, 0.268 mmol) similar to Example3 as white solid (25 mg, 41%). ¹H NMR (DMSO-d₆): 12.23 (s, 1H), 10.78(s, 1H), 7.64 (d, J=7.8 Hz, 1H), 7.11-6.92 (m, 4H), 6.41 (brs, 1H), 6.0(brs, 1H), 3.83 (s, 3H), 2.30 (s, 3H), 2.17 (s, 3H).

EXAMPLE 724-(2,4-Dimethylphenyl)-1-(4,5-dimethylfuran-2-ylmethylidne)semicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylsemicarbazide (40 mg 0.223 mmol) and4,5-dimethylfurylcarboxaldehyde (32 mg, 0.268 mmol) similar to Example 3and isolated as a white solid (12 mg, 19%). ¹H NMR (DMSO-d₆): 10.45 (s,1H), 8.03 (s, 1H), 7.59 (s, 1H), 7.44 (d, J=7.6 Hz, 1H), 6.88-6.81 (m,2H), 6.53 (s, 1H), 2.10 (s, 3H), 2.09 (s, 3H), 2.08 (s, 3H), 2.06 (s,3H).

EXAMPLE 734-(2,4-Dimethylphenyl)-1-(thiophen-2-ylmethylidene)semicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylsemicarbazide (40 mg 0.223 mmol) and2-thiophenecarboxaldehyde (30 mg, 0.268 mmol) similar to Example 3 andisolated as a white solid (10 mg, 16%). ¹H NMR (DMSO-d₆): 10.45 (s, 1H),8.18 (s, 1H), 8.14 (s, 1H), 7.72 (d, J=8.4 Hz, 1H), 7.62 (d, J=5.1 Hz,1H), 7.41 (d, J=2.7 Hz, 1H), 7.12-6.92 (m, 3H), 2.10 (s, 3H), 2.23 (s,3H), 2.22 (s, 3H).

EXAMPLE 744-(2,4-Dimethylphenyl)-1-(1-methylimidazol-2-ylmethylidene)semicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylsemicarbazide (40 mg 0.223 mmol) and1-methyl-2-imidazolecarboxaldehyde (30 mg, 0.268 mmol) similar toExample 3 and isolated as a white solid (3 mg, 5%). ¹H NMR (DMSO-d₆):10.85 (s, 1H), 8.14 (s, 1H), 7.96 (s, 1H), 7.91 (s, 1H), 7.48 (d, J=8.8Hz, 1H), 7.02-6.92 (m, 3H), 3.30 (s, 3H), 2.24 (s, 3H), 2.16 (s, 3H).

EXAMPLE 754-(2,4-Dimethylphenyl)-1-(6-methylpyridin-2-ylmethylidene)semicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylsemicarbazide (40 mg 0.223 mmol) and6-methyl-2-pyridinecarboxaldehyde (32 mg, 0.268 mmol) similar to Example3 and isolated as a white solid (15 mg, 24%). ¹H NMR (DMSO-d₆): 10.93(s, 1H), 8.55 (s, 1H), 8.02 (d, J=8.1 Hz, 1H), 7.92 (s, 1H), 7.72 (t,J=8.1 Hz, 1H), 7.45 (d, J=8.1 Hz, 1H), 7.23 (d, J=8.1 Hz, 1H), 7.04-6.95(m, 2H), 2.47 (s, 3H), 2.26 (s, 3H), 2.22 (s, 3H).

EXAMPLE 764-(2,4-Dimethylphenyl)-1-(2-hydroxy-4-methylbenzylidene)semicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylsemicarbazide (40 mg 0.223 mmol) and2-hydroxy-4-methylbenzaldehyde (36 mg, 0.268 mmol) similar to Example 3and isolated as a white solid (37 mg, 56%). ¹H NMR (DMSO-d₆): 10.64 (s,1H), 9.86 (s, 1H), 8.39 (s, 1H), 8.20 (s, 1H), 7.62 (s, 1H), 7.51 (d,J=8.0 Hz, 1H), 7.03-6.75 (m, 3H), 2.25 (s, 3H), 2.22 (s, 3H), 2.16 (s,3H).

EXAMPLE 774-(2,4-Dimethylphenyl)-1-(2-hydroxy-5-chlorobenzylidene)semicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylsemicarbazide (40 mg 0.223 mmol) and2-hydroxy-5-chlorobenzaldehyde (42 mg, 0.268 mmol) similar to Example 3and isolated as a white solid (20 mg, 28%). ¹H NMR (DMSO-d₆): 10.68 (s,1H), 8.57 (s, 1H), 8.17 (s, 1H), 7.99 (d, J=2.7 Hz, 1H), 7.38 (d, J=7.5Hz, 1H), 7.45 (dd, J=3.0, 8.4 Hz, 1H), 7.04 (s, 1H), 6.95 (d, J=7.5 Hz,1H), 6.87 (d, J=8.4 Hz, 1H), 2.26 (s, 3H), 2.12 (s, 3H).

EXAMPLE 784-(2,4-Dimethylphenyl)-1-(2,4,5-trimethoxybenzylidene)semicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylsemicarbazide (40 mg 0.223 mmol) and2-hydroxy-5-chlorobenzaldehyde (56 mg, 0.268 mmol) similar to Example 3and isolated as a white solid (45 mg, 56%). ¹H NMR (DMSO-d₆): 10.57 (s,1H), 8.45 (s, 1H), 8.19 (s, 1H), 7.61 (d, J=8.1 Hz, 1H), 7.50 (s, 1H),7.02-6.94 (m, 2H), 6.72 (s, 1H), 3.83 (s, 6H), 3.76 (s, 3H), 2.24 (s,3H), 2.18 (s, 3H).

EXAMPLE 794-(2,4-Dimethylphenyl)-1-(4-bromo-1H-pyrazol-5-ylmethylidne)semicarbazide

The title compound was prepared from a mixture of2,4-dimethylphenylsemicarbazide (40 mg 0.223 mmol) and4-bromo-1H-pyrazolo-5-carboxaldehyde (47 mg, 0.268 mmol) similar toExample 3 and isolated as a white solid (10 mg, 13%). ¹H NMR (DMSO-d₆):10.67 (s, 1H), 8.10 (s, 1H), 7.88 (d, J=8.1 Hz, 1H), 7.62-7.50 (m, 2H),7.25 (brs, 1H), 7.02-6.94 (m, 2H), 6.52 (d, J=8.1 Hz, 1H), 2.23 (s, 3H),2.18 (s, 3H).

EXAMPLE 804-(2,4-Dimethylphenyl)-1-(2-methylthiazole-5-carbonyl)semicarbazide

The title compound was prepared from a mixture2,4-dimethylphenylisocyanate (50 mg, 0.34 mmol) and2-methylthiazole-5-carboxylic acid hydrazide (59 mg, 0.37 mmol) inmethylenedichloride (10 mL). The mixture was stirred at room temperaturefor 2 h, and the resultant precipitate was collected by filtration andisolated as pale white solid (66 mg, 64%). ¹H NMR (DMSO-d₆): 10.00 (s,1H), 8.39 (s, 1H), 8.25 (s, 1H), 7.88 (s, 1H), 7.46 (d, J=8.1 Hz, 1H),6.99 (s, 1H), 6.87 (d, J=8.1 Hz, 1H), 2.72 (s, 3H), 2.22 (s, 3H), 2.16(s, 3H).

EXAMPLE 81 4-(2,4-Dimethylphenyl)-1-(4-chlorobenzoyl)semicarbazide

The title compound was prepared from a mixture2,4-dimethylphenylisocyanate (50 mg, 34 mmol) and 4-chlorobenzoic acidhydrazide (63 mg, 37 mmol) in methylenedichloride (10 ml). The mixturewas stirred at room temperature for 2 h and the resultant precipitatewas collected by filtration and isolated as pale white solid (90 mg,89%). ¹H NMR (DMSO-d₆): 10.39 (s, 1H), 8.34 (s, 1H), 8.05 (s, 1H), 7.94(d, J=6.6 Hz, 1H), 7.57 (d, J=6.6 Hz, 1H), 7.44 (d, J=7.5 Hz, 1H), 6.96(s, 1H), 6.92 (d, J=7.5 Hz, 1H), 2.72 (s, 3H), 2.22 (s, 3H), 2.16 (s,3H).

EXAMPLE 824-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(2-methylthiazole-5-carbonyl)thiosemicarbazide

The title compound was prepared from a mixture ofbicyclo[2.2.1]hept-5-en-2-ylisothiocyanate (50 mg, 0.33 mmol) and2-methylthiazole-5-carboxylic acid hydrazide (58 mg, 0.36 mmol) similarto Example 80 and isolated as a white solid (10 mg, 9%). ¹H NMR(DMSO-d₆): 10.11 (s, 1H), 9.34 (s, 1H), 8.22 (s, 1H), 7.73 (s, 1H), 6.07(ddd, J=8.1, 5.1, 2.7 Hz, 2H), 3.99 (s, 1H), 2.82 (brs, J=8.8 Hz, 2H),2.72 (s, 3H), 1.52-1.35 (m, 4H).

EXAMPLE 834-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-chlorobenzoyl)thiosemicarbazide

The title compound was prepared from a mixture ofbicyclo[2.2.1]hept-5-en-2-ylisothiocyanate (50 mg, 0.33 mmol) and4-chlorophenylhydrazide (61 mg, 0.36 mmol) similar to Example 80 andisolated as a white solid (60 mg, 56%). ¹H NMR (DMSO-d₆): 10.37 (s, 1H),9.28 (s, 1H), 7.93 (d, J=8.8 Hz, 2H), 7.56 (d, J=8.8 Hz, 2H), 6.02 (ddd,J=8.1, 5.1, 2.7 Hz, 2H), 4.08 (s, 1H), 2.83 (brs, J=8.8 Hz, 2H), 2.72(s, 3H), 1.52-1.35 (m, 4H).

EXAMPLE 844-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(3-hydroxybenzoyl)thiosemicarbazide

The title compound was prepared from a mixture ofbicyclo[2.2.1]hept-5-en-2-ylisothiocyanate (50 mg, 0.33 mmol) and3-hydroxyphenylhyrdazide (55 mg, 0.36 mmol) similar to Example 80 andisolated as a white solid (10 mg, 10%). ¹H NMR (DMSO-d₆): 10.16 (s, 1H),9.69 (s, 1H), 9.28 (s, 1H), 7.35-7.03 (m, 3H), 6.95 (d, J=7.5 Hz, 1H),6.10 (ddd, J=8.1, 5.1, 2.7 Hz, 2H), 4.02 (s, 1H), 2.83 (brs, J=8.8 Hz,2H), 2.72 (s, 3H), 1.52-1.35 (m, 4H).

EXAMPLE 854-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4,5-dimethylfuran-2-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (100 mg, 0.6mmol) and 4,5-dimethylfurylaldehyde (87 mg, 0.72 mmol) similar toExample 3 and isolated as an off white solid (25 mg, 14%). ¹H NMR(CDCl₃): 9.48 (s, 1H), 7.55 (s, 1H), 7.35 (brs, 1H), 6.51 (s, 1H), 6.16(ddd, J=8.1, 5.1, 2.7 Hz, 2H), 4.22 (m, 1H), 3.03 (s, 1H), 2.93 (s, 1H),2.27 (s, 3H), 1.96 (s, 3H), 1.85 (m, 1H), 1.64(m, 1H), 1.54(m, 1H), 1.41(m, 1H).

EXAMPLE 864-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(2-methoxy-4-dimethylaminobenzylidene)thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (100 mg, 0.6mmol) and 2-methoxy-4-dimethylaminobenzaldehyde (97 mg, 0.72 mmol)similar to Example 3 and isolated as a yellow solid (60 mg, 29%). ¹H NMR(CDCl₃): 9.08 (s, 1H), 8.05 (s, 1H), 7.65 (brs, 1H), 7.45 (brs, 1H),6.27 (brs, 1H), 6.22 (ddd, J=8.1, 5.1, 2.7 Hz, 2H), 6.00 (s, 1H), 4.32(m, 1H), 3.82 (s, 3H), 3.03 (s, 6H), 2.93 (s, 1H), 1.85 (m, 1H), 1.64(m, 1H), 1.54 (m, 1H), 1.41 (m, 1H).

EXAMPLE 874-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(1-methylpyrrol-2-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (100 mg, 0.6mmol) and 6-methyl-2-pyrrolecarboxaldehyde (78 mg, 0.72 mmol) similar toExample 3 and isolated as a yellow solid (43 mg, 26%). ¹H NMR (CDCl₃):9.08 (s, 1H), 7.20 (d, J=7.5 Hz, 1H), 6.75 (t, J 5.1 Hz, 1H), 6.61 (t,J=5.1 Hz, 1H), 6.27 (brs, 2H), 6.22-6.12 (d, 3H), 4.26 (t, J=75 Hz, 1H),3.80 (s, 3H), 2.90 (s, 1H), 2.85 (s, 1H), 1.93-1.85 (m, 1H), 1.64-1.54(m, H), 1.41-1.24 (m, 2H).

EXAMPLE 884-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-pyrrolidinobenzylidene)thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (100 mg, 0.6mmol) and 4-pyrrolidinobenzaldehyde (126 mg, 0.72 mmol) similar toExample 3 and isolated as a white solid (60 mg, 29%). ¹H NMR (CDCl₃):9.01 (s, 1H), 7.65 (s, 1H), 7.50-7.26 (m, 2H), 6.52 (d, J=8.8 Hz, 2H),6.16 (ddd, J=8.1, 5.1, 2.7 Hz, 2H), 4.29 (t, J=8.1 Hz, 1H), 3.36-3.32(m, 4H), 3.04 (s, 1H), 2.95 (s, 1H), 2.07-1.99 (m, 4H ), 1.87 (ddd,J=7.6, 7.0, 2.7 Hz, 1H), 1.63 (m, 1H), 1.53-1.38 (m, 2H).

EXAMPLE 894-(4-Bicyclo[2.2.1]hept-5-en-2-yl)-1-(6-bromopyridin-2-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (100 mg, 0.6mmol) and 6-bromo-2-pyridenylcarboxaldehyde (133 mg, 0.72 mmol) similarto Example 3 and isolated as a white solid (80 mg, 38%). ¹H NMR (CDCl₃):9.45 (s, 1H), 7.81 (s, 1H), 7.79 (dd, J=7.5, 0.6 Hz, 2H), 4.60 (d, J=7.5Hz, 2H), 7.49-7.26 (m, 2H), 6.21 (ddd, J=8.1, 35.1, 2.7 Hz, 2H), 4.26(t, J=7.6 Hz, 1H), 3.06 (s, 1H), 2.96 (s, 1H), 2.07-1.99 (m, 4H), 1.92(ddd, J=7.6, 7.0, 2.4 Hz, 1H), 1.67(m, 1H), 1.51-1.40 (m, 2H).

EXAMPLE 904-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(1,2,3-thiadiazol-4-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (35 mg, 0.21mmol) and 1,2,3-thiadiazole-4-carboxaldehyde (33mg, 0.287 mmol) similarto Example 3 and isolated as a white solid (47 mg, 80%). ¹H NMR (CDCl₃):9.95 (s, 1H), 8.85 (s, 1H), 7.70 (brs, 1H), 6.16 (ddd, J=8.1, 5.1, 2.7Hz, 2H), 4.27 (t, J=8.7 Hz, 1H), 3.06 (s, 1H), 2.96 (s, 1H), 1.92 (ddd,J=7.6, 7.0, 2.4 Hz, 1H), 1.67 (m, 1H), 1.51-1.40 (m, 2H).

EXAMPLE 914-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-morpholino-3-nitrobenzylidene)thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (35 mg, 0.21mmol) and 4-morpholino-3-nitrobenzaldehyde (68 mg, 0.287 mmol) similarto Example 3 and isolated as a yellow solid (42 mg, 52%). ¹H NMR(CDCl₃): 9.91 (s, 1H), 7.90 (d, J=2.1 Hz, 1H), 7.22 (s, 1H), 7.58 (dd,J=8.8, 2.4 Hz, 1H), 7.18 (d, J=2.4 Hz, 1H), 7.01 (d, J=8.8 Hz, 1H), 6.10(ddd, J=8.1, 5.1, 2.7 Hz, 2H), 4.27 (t, J=8.7 Hz, 1H), 3.75-3.72 (m,4H), 3.02-2.99 (m, 4H), 2.93 (s, 1H), 2.85 (s, 1H), 1.82 (ddd, J=7.6,7.0, 2.4 Hz, 1H), 1.57 (m, 1H), 1.39-1.28 (m, 2H).

EXAMPLE 924-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-bromo-1H-pyrazol-5-ylmethylidne)thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (35 mg, 0.21mmol) and 4-bromo-1H-pyrazol-5-carboxaldehyde (50 mg, 0.287 mmol)similar to Example 3 and isolated as a white solid (45 mg, 63%). ¹H NMR(CDCl₃): 11.85 (s, 1H), 8.04-8.00 (m, 3H), 6.10 (ddd, J=8.1, 5.1, 2.7Hz, 2H), 4.27 (t, J=8.7 Hz, 1H), 2.93 (m, 2H), 1.82 (m, 2H), 1.57-1.3(m, 3H).

EXAMPLE 934-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-chloro-1-methylpyrazol-3-ylmethylidene)thiosemicarbazide

The title compound was prepared from a mixture ofN1-bicyclo[2.2.1]hept-5-en-2-ylhydrazine-1-carbothioamide (35 mg, 0.21mmol) and 4-chloro-1-methylpyrazole-3-carboxaldehyde (41 mg, 0.287 mmol)similar to Example 3 and isolated as a white solid (15 mg, 24%). ¹H NMR(CDCl₃): 11.91 (s, 1H), 7.24 (s, 1H), 7.45 (s, 1H), 7.35 (s, 1H), 6.10(ddd, J=8.1, 5.1, 2.7 Hz, 2H), 4.26 (t, J=7.5 Hz, 1H), 3.99 (s, 3H),3.02 (s, 1H), 2.93 (s, 1H), 1.88 (ddd, J=8.4, 7.0, 2.7 Hz, 1H), 1.61 (m,1H), 1.52-1.38 (m, 2H).

EXAMPLE 94 Identification of4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridin-2-ylmethylidene)thiosemicarbazideand Analogs as Caspase Cascade Activators and Inducers of Apoptosis inSolid Tumor Cells

Human breast cancer cell lines T47D and ZR-75-1 were grown according tomedia component mixtures designated by American Type CultureCollection+10% FCS (Invitrogen Corporation), in a 5% CO₂-95% humidityincubator at 37° C. T-47D and ZR-75-1 cells were maintained at a celldensity between 50 and 80% confluency at a cell density of 0.1 to0.6×10⁶ cells/mL. Cells were harvested at 600×g and resuspended at0.65×10⁶ cells/mL into appropriate media+10% FCS. An aliquot of 45 μL ofcells was added to a well of a 96-well microtiter plate containing 2.5μL of a 10% DMSO in RPMI-1640 media solution containing 0.16 to 100 μMof4-(bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridin-2-ylmethylidene)thiosemicarbazideor other test compound (0.016 to 10 μM final). An aliquot of 22.5 μL ofcells was added to a well of a 384-well microtiter plate containing 2.5μL of a 10% DMSO in RPMI-1640 media solution without test compound asthe control sample. The samples were mixed by agitation and thenincubated at 37° C. for 48 h in a 5% CO₂-95% humidity incubator. Afterincubation, the samples were removed from the incubator and 25 μL of asolution containing 14 μM of N-(Ac-DEVD)-N′-ethoxycarbonyl-R110 (SEQ IDNO:1) fluorogenic substrate (Maxim Pharmaceuticals, Inc.; WO99/18856),20% sucrose (Sigma), 20 mM DTT (Sigma), 200 mM NaCl (Sigma), 40 mM NaPIPES buffer pH 7.2 (Sigma), and 500 μg/ml lysolecithin (Calbiochem) wasadded. The samples were mixed by agitation and incubated at roomtemperature. Using a fluorescent plate reader (Model SpectraMax Gemini,Molecular Devices), an initial reading (T=0) was made approximately 1-2min after addition of the substrate solution, employing excitation at485 nm and emission at 530 nm, to determine the background fluorescenceof the control sample. After the 3 h incubation, the samples were readfor fluorescence as above (T=3 h).

Calculation:

The Relative Fluorescence Unit values (RFU) were used to calculate thesample readings as follows:

RFU _((T=3 h))−Control RFU _((T=0))=Net RFU_((T=3 h))

The activity of caspase cascade activation was determined by the ratioof the net RFU value for4-(bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridin-2-ylmethylidene)thiosemicarbazideor other test compound to that of control samples. The EC₅₀ (nM) wasdetermined by a sigmoidal dose-response calculation (Prism 2.0, GraphPadSoftware Inc.). The caspase activity (Ratio) and potency (EC₅₀) aresummarized in Table I:

TABLE I Caspase Activity and Potency T-47D ZR-75-1 EC₅₀ EC₅₀ ExampleRatio (nM) Ratio (nM) 16 3.5 360 3.0 862 17 3.3 866 2.5 INACTIVE

Thus,4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridin-2-ylmethylidene)thiosemicarbazideis identified as a potent caspase cascade activator and inducer ofapoptosis in solid tumor cells.

EXAMPLE 95 Identification of4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridin-2-ylmethylidene)thiosemicarbazideas Antineoplastic Compound that Inhibits Cell Proliferation (GI₅₀)

All the cell lines were grown according to the conditions recommended byAmerican Type Culture Collection. An aliquot of 90 μL of cells (2.2×10⁴cells/mL) was added to a well of a 96-well microtiter plate containing10 μL of a 10% DMSO in RPMI-1640 media solution containing 1 nM to 100μM of4-(bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridin-2-ylmethylidene)thiosemicarbazide(0.1 nM to 10 μM final). An aliquot of 90 μL of cells was added to awell of a 96-well microtiter plate containing 10 μL of a 10% DMSO inRPMI-1640 media solution without compound as the control sample formaximal cell proliferation (A_(Max)). The samples were mixed byagitation and then incubated at 37° C. for 48 h in a 5% CO₂-95% humidityincubator. After incubation, the samples were removed from the incubatorand 20 μL of CellTiter 96 AQ_(UEOUS) One Solution Cell Proliferation™reagent (Promega) was added. The samples were mixed by agitation andincubated at 37° C. for 2-4 h in a 5% CO₂-95% humidity incubator. Usingan absorbance plate reader (Model 1420 Wallac Instruments), an initialreading (T=0) was made approximately 1-2 min after addition of thesolution, employing absorbance at 490 nm. This determines the possiblebackground absorbance of the test compounds. No absorbance for4-(bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridin-2-ylmethylidene)thiosemicarbazidewas found at 490 nm. After the 2-4 h incubation, the samples were readfor absorbance as above (A_(Test)).

Baseline for GI₅₀ (dose for 50% inhibition of cell proliferation) ofinitial cell numbers were determined by adding an aliquot of 90 μL ofcells or 90 μL of media, respectively, to wells of a 96-well microtiterplate containing 10 μL of a 10% DMSO in RPMI-1640 media solution. Thesamples were mixed by agitation and then incubated at 37° C. for 0.5 hin a 5% CO₂-95% humidity incubator. After incubation, the samples wereremoved from the incubator and 20 μL of CellTiter 96 AQ_(UEOUS) OneSolution Cell Proliferation™ reagent (Promega) was added. The sampleswere mixed by agitation and incubated at 37° C. for 24 h in a 5% CO₂-95%humidity incubator. Absorbance was read as above, (A_(Start)) definingabsorbance for initial cell number used as baseline in GI₅₀determinations.

Calculation:

GI₅₀ (dose for 50% inhibition of cell proliferation) is theconcentration where [(A_(Test)−A_(Start))/(A_(Max)−A_(Start))] =0.5.

The GI₅₀ (nM) are summarized in Table II:

TABLE II GI₅₀ in Cancer Cells GI₅₀ (nM) MDA- T47- MCF- SK-Br- MB- PC-DLD- NCI- Compound D 7 3 231 3 HeLa 1 H23 K562 Example 16 5 7 5 30 30 6090 30 50 Example 17 1000 300 750 ND ND ND 1700 3000 3000 ND = not done

Thus,4-(bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridin-2-ylmethylidene)thiosemicarbazideis identified as antineoplastic compound that inhibits cellproliferation in a variety of cancer cell lines.

EXAMPLE 96 Identification of4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridin-2-ylmethylidene)thiosemicarbazideas Antineoplastic Compound that Inhibits Cell Proliferation (GI₅₀) ofDrug Resistant Cancer Cells

Cell lines P388 and P388/ADR were grown according to the conditionsrecommended by American Type Culture Collection. The cell proliferationassay and the calculation of GI₅₀s were performed as described inExample 95.

The GI₅₀ (nM) in drug sensitive (P388) and drug resistant (P388/ADR)cell lines are summarized in Table III.

TABLE III GI₅₀ in P388 and P388/ADR cell lines GI₅₀(nM) Compound P388P388/ADR Example 16 20 20 Example 17 ND ND ND = not done

Thus,4-(bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridin-2-ylmethylidene)thiosemicarbazideis identified as an antineoplastic compound that inhibits cellproliferation in both drug-sensitive and drug-resistant cell lines.

Having now fully described this invention, it will be understood bythose of ordinary skill in the art that the same can be performed withina wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or anyembodiment thereof. All patents, patent applications and publicationscited herein are fully incorporated by reference herein in theirentirety.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 1 <210> SEQ ID NO 1 <211> LENGTH: 4<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Fluorogenic substrate <400> SEQUENCE: 1Asp Glu Val Asp 1

What is claimed is:
 1. A pharmaceutical composition, comprising acompound selected from the group consisting of:4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-benzylidene-thiosemicarbazide;4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridin-4-ylmethylidene)thiosemicarbazide;4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyrrol-2-ylmethylidene)thiosemicarbazide;4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-hydroxybenzylidene)thiosemicarbazide;4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridin-3-ylmethylidene)thiosemicarbazide;4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(6-methyl-pyridin-2-ylmethylidene)thiosemicarbazide;4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(pyridine-2-carbonyl)thiosemicarbazide;4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(thiazol-2-ylethylidene)thiosemicarbazide;4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4,5-dimethylfuran-2-ylmethylidene)thiosemicarbazide;4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(2-methoxy-4-dimethylaminobenzylidene)thiosemicarbazide;4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(1-methylpyrrol-2-ylmethylidene)thiosemicarbazide;4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-pyrrolidinobenzylidene)thiosemicarbazide;4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(6-bromopyridin-2-ylmethylidene)thiosemicarbazide;4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(1,2,3-thiadiazol-4-ylmethylidene)thiosemicarbazide;4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-morpholino-3-nitrobenzylidene)thiosemicarbazide;4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-bromo-1H-pyrazol-5-ylmethylidne)thiosemicarbazide;4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(4-chloro-1-methylpyrazol-3-ylmethylidene)thiosemicarbazide;and4-(Bicyclo[2.2.1]hept-5-en-2-yl)-1-(2-methylthiazole-5-carbonyl)thiosemicarbazide;and a pharmaceutically acceptable carrier.
 2. The pharmaceuticalcomposition of claim 1, further comprising at least one known cancerchemotherapeutic agent, or a pharmaceutically acceptable salt of saidagent.
 3. The pharmaceutical composition of claim 2, wherein said knowncancer chemotherapeutic agent is selected from the group consisting ofbusulfan, cis-platin, mitomycin C, carboplatin, colchicine, vinblastine,paclitaxel, docetaxel, camptothecin, topotecan, doxorubicin, etoposide,5-azacytidine, 5-fluorouracil, methotrexate, 5-fluoro-2′-deoxy-uridine,ara-C, hydroxyurea, thioguanine, melphalan, chlorambucil,cyclophosamide, ifosfamide, vincristine, mitoguazone, epinibicin,aclanibicin, bleomycin, mitoxantrone, elliptinium, fludarabine,octreotide, retinoic acid, tamoxifen, campath, imatinib mesylate,tratuzumab, rituximab and alanosine.
 4. The pharmaceutical compositionof any one of claims 1-3, wherein said compound is4-(bicyclo[2.2.1]hept-5-en-2-yl)-1-(6-methyl-pyridin-2-ylmethylidene)thiosemicarbazide.5. A pharmaceutical composition, comprising a compound selected from thegroup consisting of:4-(2,4-Dimethylphenyl)-1-benzylidene-thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(6-methyl-pyridin-2-ylmethylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(2-nitrobenzylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(3chlorobenzylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(2-chlorobenzylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(3-hydroxybenzylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(2-hydroxybenzylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(4-dimethylaminobenzylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(furan-2-ylmethylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(thiophen-2-ylmethylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(4,5-dimethylfuran-2-ylmethylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(N-methylpyrrol-2-ylmethylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(5-nitrothiophen-2-ylmethylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(imidazol-2-ylmethylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(6-bromo-pyridin-2-ylmethylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(4-bromo-1H-pyrazol-5-ylmethylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(4-chloro-1-methylpyrazol-3-ylmethylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(1,2,3-thiazol-4-ylmethylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(thiazol-2-ylethylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(2-furan-2-ylmethylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(3-chlorobenzylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(benzylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(4-pyrrolidinobenzylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(pyridin-2-ylmethylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(2-methoxy-4-dimethylaminobenzylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(4-chloro-1-methylpyrazol-3-ylmethylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(3-hydroxybenzylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(1-methylpyrrol-2-ylmethylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(4,5-dimethylfuran-2-ylmethylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(thiophene-2-ylmethylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(6-methylpyridin-2-ylmethylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(2-hydroxy-4-methylbenzylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(2-hydroxy-5-chlorobenzylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(4-bromo-1H-pyrazol-5-ylmethylidne)semicarbazide;4-(2,4-Dimethylphenyl)-1-(2-methylthiazole-5-carbonyl)semicarbazide; and4-(2,4-Dimethylphenyl)-1-(4-chlorobenzoyl)semicarbazide, and apharmaceutically acceptable carrier.
 6. The pharmaceutical compositionof claim 5, further comprising at least one known cancerchemotherapeutic agent, or a pharmaceutically acceptable salt of saidagent.
 7. The pharmaceutical composition of claim 6, wherein said knowncancer chemotherapeutic agent is selected from the group consisting ofbusulfan, cis-platin, mitomycin C, carboplatin, colchicine, vinblastine,paclitaxel, docetaxel, camptothecin, topotecan, doxorubicin, etoposide,5-azacytidine, 5-fluorouracil, methotrexate, 5-fluoro-2′-deoxy-uridine,ara-C, hydroxyurea, thioguanine, melphalan, chlorambucil,cyclophosamide, ifosfamide, vincristine, mitoguazone, epirubicin,aclarubicin, bleomycin, mitoxantrone, elliptinium, fludarabine,octreotide, retinoic acid, tamoxifen, campath, imatinib mesylate,tratuzumab, rituximab and alanosine.
 8. The pharmaceutical compositionof any one of claims 5-7, wherein said compound is4-(2,4-Dimethylphenyl)-1-benzylidene-thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(2-nitrobenzylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(3-chlorobenzylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(2-chlorobenzylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(3-hydroxybenzylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(2-hydroxybenzylidene)thiosemicarbazide; or4-(2,4-Dimethylphenyl)-1-(4-dimethylamino-benzylidene)thiosemicarbazide.9. The pharmaceutical composition of any one of claims 5-7, wherein saidcompound is 4-(2,4-Dimethylphenyl)-1-(3-chlorobenzylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(benzylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(3-hydroxybenzylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(2-hydroxy-4-methyl-benzylidene)semicarbazide;or4-(2,4-Dimethylphenyl)-1-(2-hydroxy-5-chloro-benzylidene)semicarbazide.10. The pharmaceutical composition of any one of claims 5-7, whereinsaid compound is4-(2,4-Dimethylphenyl)-1-(6-methyl-pyridin-2-ylmethylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(furan-2-ylmethylidine)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(thiophen-2-ylmethylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(4,5-dimethylfuran-2-ylmethylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(imidazol-2-ylmethylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(6-bromo-pyridin-2-ylmethylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(4-bromo-1H-pyrazol-5-ylmethylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(4-chloro-1-methylpyrazol-3-ylmethylidene)thiosemicarbazide;4-(2,4-Dimethylphenyl)-1-(1,2,3-thiazol-4-ylmethylidene)thiosemicarbazide;or 4-(2,4-Dimethylphenyl)-1-(thiazol-2-ylethylidene)thiosemicarbazide.11. The pharmaceutical composition of any one of claims 5-7, whereinsaid compound is4-(2,4-Dimethylphenyl)-1-(2-furan-2-ylmethylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(pyridin-2-ylmethylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(4-chloro-1-methylpyrazol-3-ylmethylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(1-methylpyrrol-2-ylmethylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(4,5-dimethylfuran-2-ylmethylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(thiophene-2-ylmethylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(6-methylpyridin-2-ylmethylidene)semicarbazide;4-(2,4-Dimethylphenyl)-1-(4-bromo-1H-pyrazol-5-ylmethylidne)semicarbazide;4-(2,4-Dimethylphenyl)-1-(2-methylthiazole-5-carbonyl)semicarbazide; or4-(2,4-Dimethylphenyl)-1-(4-chlorobenzoyl)semicarbazide.